Methods and Compositions for the Treatment of Gastrointestinal Disorders

ABSTRACT

Compositions and related methods for treating IBS and other gastrointestinal disorders and conditions (e.g., gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), duodenogastric reflux, Crohn&#39;s disease, ulcerative colitis, inflammatory bowel disease, functional heartburn, dyspepsia (including functional dyspepsia or nonulcer dyspepsia), gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudoobstruction), and disorders and conditions associated with constipation, e.g., constipation associated with use of opiate pain killers, post-surgical constipation, and constipation associated with neuropathic disorders as well as other conditions and disorders are described. The compositions feature peptides that activate the guanylate cyclase C (GC-C) receptor.

PRIORITY CLAIM

This application is a continuation of, and claims priority under 35U.S.C. §120 to U.S. patent application Ser. No. 14/024,552 filed Sep.11, 2013, which is a continuation of U.S. patent application Ser. No.13/427,431 filed Mar. 22, 2012, which is a continuation of U.S. patentapplication Ser. No. 13/291,526 filed Nov. 8, 2011, which is acontinuation of U.S. patent application Ser. No. 12/568,107 filed Sep.28, 2009, which is a continuation of U.S. patent application Ser. No.10/868,744 filed Jun. 14, 2004 and also claims the benefit of priorityunder 35 U.S.C. §119 (e) to United States Provisional Application Nos.60/478,492, filed Jun. 13, 2003; 60/532,361 filed Dec. 23, 2003; and60/571,386 filed May 14, 2004. The disclosure of the prior applicationsis considered part of (and is incorporated by reference in) thedisclosure of this application.

TECHNICAL FIELD

This invention relates to methods and compositions for treatinggastrointestinal disorders, obesity, congestive heart failure, benignprostatic hyperplasia and other disorders.

SEQUENCE LISTING

This application incorporates by reference in its entirety the SequenceListing entitled 14184-034001.txt, containing 2,417 kilobytes of data,created Sep. 1, 2006, and filed with parent application U.S. patentapplication Ser. No. 10/868,744 on Sep. 11, 2006 and incorporated inU.S. patent application Ser. No. 12/568,107 in computer readable-format(CRF) and electronic .txt format.

BACKGROUND

Irritable bowel syndrome (IBS) is a common chronic disorder of theintestine that affects 20 to 60 million individuals in the US alone(Lehman Brothers, Global Healthcare-Irritable Bowel Syndrome IndustryUpdate, September 1999). IBS is the most common disorder diagnosed bygastroenterologists (28% of patients examined) and accounts for 12% ofvisits to primary care physicians (Camilleri 2001 Gastroenterology120:652-668). In the US, the economic impact of IBS is estimated at $25billion annually, through direct costs of health care use and indirectcosts of absenteeism from work (Talley 1995 Gastroenterology109:1736-1741). Patients with IBS have three times more absenteeism fromwork and report a reduced quality of life. Sufferers may be unable orunwilling to attend social events, maintain employment, or travel evenshort distances (Drossman 1993 Dig Dis Sci 38:1569-1580). There is atremendous unmet medical need in this population since few prescriptionoptions exist to treat IBS.

Patients with IBS suffer from abdominal pain and a disturbed bowelpattern. Three subgroups of IBS patients have been defined based on thepredominant bowel habit: constipation-predominant (c-IBS),diarrhea-predominant (d-IBS) or alternating between the two (a-IBS).Estimates of individuals who suffer from c-IBS range from 20-50% of theIBS patients with 30% frequently cited. In contrast to the other twosubgroups that have a similar gender ratio, c-IBS is more common inwomen (ratio of 3:1) (Talley et al. 1995 Am J Epidemiol 142:76-83).

The definition and diagnostic criteria for IBS have been formalized inthe “Rome Criteria” (Drossman et al. 1999 Gut 45:Suppl II:1-81), whichare well accepted in clinical practice. However, the complexity ofsymptoms has not been explained by anatomical abnormalities or metabolicchanges. This has led to the classification of IBS as a functional GIdisorder, which is diagnosed on the basis of the Rome criteria andlimited evaluation to exclude organic disease (Ringel et al. 2001 AnnuRev Med 52: 319-338). IBS is considered to be a “biopsychosocial”disorder resulting from a combination of three interacting mechanisms:altered bowel motility, an increased sensitivity of the intestine orcolon to pain stimuli (visceral sensitivity) and psychosocial factors(Camilleri 2001 Gastroenterology 120:652-668). Recently, there has beenincreasing evidence for a role of inflammation in the etiology of IBS.Reports indicate that subsets of IBS patients have small but significantincreases in colonic inflammatory and mast cells, increased induciblenitric oxide (NO) and synthase (iNOS) and altered expression ofinflammatory cytokines (reviewed by Talley 2000, Medscape Coverage ofDDW Week).

SUMMARY OF THE INVENTION

The present invention features compositions and related methods fortreating IBS and other gastrointestinal disorders and conditions (e.g.,gastrointestinal motility disorders, functional gastrointestinaldisorders, gastroesophageal reflux disease (GERD), duodenogastricreflux, Crohn's disease, ulcerative colitis, inflammatory bowel disease,functional heartburn, dyspepsia (including functional dyspepsia ornonulcer dyspepsia), gastroparesis, chronic intestinalpseudo-obstruction (or colonic pseudoobstruction), and disorders andconditions associated with constipation, e.g., constipation associatedwith use of opiate pain killers, post-surgical constipation, andconstipation associated with neuropathic disorders as well as otherconditions and disorders. The compositions feature peptides thatactivate the guanylate cyclase C (GC-C) receptor.

The present invention also features compositions and related methods fortreating obesity, congestive heart failure and benign prostatichyperplasia (BPH).

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are useful because theycan increase gastrointestinal motility.

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are useful, in part,because they can decrease inflammation.

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are also useful becausethey can decrease gastrointestinal pain or visceral pain.

The invention features pharmaceutical compositions comprising certainpeptides that are capable of activating the guanylate-cyclase C (GC-C)receptor. Also within the invention are pharmaceutical compositionscomprising a peptide of the invention as well as combinationcompositions comprising a peptide of the invention and one or moreadditional therapeutic agents, e.g., an agent for treating constipation(e.g., a chloride channel activator such as SPI-0211; SucampoPharmaceuticals, Inc.; Bethesda, Md., a laxative such as MiraLax;Braintree Laboratories, Braintree Mass.) or some other gastrointestinaldisorder. Examples of additional therapeutic agents include: acidreducing agents such as proton pump inhibitors (e.g. omeprazole,esomeprazole, lansoprazole, pantorazole and rabeprazole), H2 receptorblockers (e.g., cimetidine, ranitidine, famotidine and nizatidine),pro-motility agents such as motilin agonists (e.g., GM-611 or mitemcinalfumarate), 5HT receptor agonists (e.g. 5HT4 receptor agonists such asZelnorm®; 5HT3 receptor agonists such as MKC-733), 5HT receptorantagonists (e.g., 5HT1, 5HT2, 5HT3 (e.g., alosetron), 5HT4 receptorantagonists, muscarinic receptor agonists, anti-inflammatory agents,antispasmodics, antidepressants, centrally-acting analgesic agents suchas opioid receptor agonists, opioid receptor antagonists (e.g.,naltrexone), agents for the treatment of Inflammatory bowel disease,Crohn's disease and ulcerative colitis (e.g., Traficet-EN™(ChemoCentryx, Inc.; San Carlos, Calif.)), agents that treatgastrointestinal or visceral pain, and cGMP phosphodiesterase inhibitors(e.g., motapizone, zaprinast, and suldinac sulfone). The peptides of theinvention can also be used in combination with agents such as tianeptine(Stablon®) and other agents described in U.S. Pat. No. 6,683,072, (E)-4(1,3bis(cyclohexylmethyl)-1,2,3,4,-tetrahydro-2,6-diono-9H-purin-8-yl)cinnamicacid nonaethylene glycol methyl ether ester and related compoundsdescribed in WO 02/067942. The peptides can also be used in combinationwith treatments entailing the administration of microorganisms useful inthe treatment of gastrointestinal disorders such as IBS. Probactrix®(The BioBalance Corporation; New York, N.Y.) is one example of aformulation that contains microorganisms useful in the treatment ofgastrointestinal disorders. The peptides can also be used in combinationwith purgatives that draw fluids to the intestine (e.g., Visicol®, acombination of sodium phosphate monobasic monohydrate and sodiumphosphate dibasic anhydrate.

In addition, the pharmaceutical compositions can include one or moreagents selected from the group consisting of: Ca channel blockers (e.g.,ziconotide), complete or partial 5HT receptor antagonists (for example5HT3 (e.g., alosetron, ATI-7000; Aryx Thearpeutics, Santa Clara Calif.),5HT4, 5HT2, and 5HT1 receptor antagonists), complete or partial 5HTreceptor agonists including 5HT3, 5HT2, 5HT4 (e.g., tegaserod, mosaprideand renzapride), 5HT1 receptor agonists, CRF receptor agonists(NBI-34041), β-3 adrenoreceptor agonists, opioid receptor agonists(e.g., loperamide, fedotozine, and fentanyl, naloxone, naltrexone,methyl nalozone, nalmefene, cypridime, beta funaltrexamine,naloxonazine, naltrindole, and norbinaltorphimine, morphine,diphenyloxylate, enkephalin pentapeptide, asimadoline, and trimebutine),NK1 receptor antagonists (e.g., ezlopitant and SR-14033), CCK receptoragonists (e.g., loxiglumide), NK1 receptor antagonists, NK3 receptorantagonists (e.g., talnetant, osanetant (SR-142801), SSR-241586),norepinephrine-serotonin reuptake inhibitors (NSRI; e.g., milnacipran),vanilloid and cannabanoid receptor agonists (e.g., arvanil), sialorphin,sialorphin-related peptides comprising the amino acid sequence QHNPR(SEQ ID NO:85) for example, VQHNPR (SEQ ID NO:86); VRQHNPR (SEQ IDNO:87); VRGQHNPR (SEQ ID NO:88); VRGPQHNPR (SEQ ID NO:89); VRGPRQHNPR(SEQ ID NO:90); VRGPRRQHNPR (SEQ ID NO:91); and RQHNPR (SEQ ID NO:92),compounds or peptides that are inhibitors of neprilysin, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1), loperamide, Tyr-Arg(kyotorphin), CCK receptor agonists (caerulein), conotoxin peptides,peptide analogs of thymulin, loxiglumide, dexloxiglumide (the R-isomerof loxiglumide) (WO 88/05774). These peptides and compounds can beadministered with the peptides of the invention (simultaneously orsequentially). They can also be covalently linked to a peptide of theinvention to create therapeutic conjugates.

The invention includes methods for treating various gastrointestinaldisorders by administering a peptide that acts as a partial or completeagonist of the GC-C receptor. The peptide contains up to four cysteinesthat form one or two disulfide bonds. In certain embodiments thedisulfide bonds are replaced by other covalent cross-links and in somecases the cysteines are substituted by other residues to provide foralternative covalent cross-links. The peptides may also include at leastone trypsin or chymotrypsin cleavage site and/or a carboxy-terminalanalgesic peptide or small molecule, e.g., AspPhe or some otheranalgesic peptide. When present within the peptide, the analgesicpeptide or small molecule may be preceded by a chymotrypsin or trypsincleavage site that allows release of the analgesic peptide or smallmolecule. The peptides and methods of the invention are also useful fortreating pain and inflammation associated with various disorders,including gastrointestinal disorders. Certain peptides include afunctional chymotrypsin or trypsin cleavage site located so as to allowinactivation of the peptide upon cleavage. Certain peptides having afunctional cleavage site undergo cleavage and gradual inactivation inthe digestive tract, and this is desirable in some circumstances. Incertain peptides, a functional chymotrypsin site is altered, increasingthe stability of the peptide in vivo(e.g., guanylin).

The invention includes methods for treating other disorders such ascongestive heart failure and benign prostatic hyperplasia byadministering a peptide or small molecule (parenterally or orally) thatacts as an agonist of the GC-C receptor. Such agents can be used incombination with natriuretic peptides (e.g., atrial natriuretic peptide,brain natriuretic peptide or C-type natriuretic peptide), a diuretic, oran inhibitor of angiotensin converting enzyme.

The invention features methods and compositions for increasingintestinal motility. Intestinal motility involves spontaneouscoordinated distentions and contractions of the stomach, intestines,colon and rectum to move food through the gastrointestinal tract duringthe digestive process.

The peptide can contain additional carboxy terminal or amino terminalamino acids or both. For example, the peptide can include an aminoterminal sequence that facilitates recombinant production of the peptideand is cleaved prior to administration of the peptide to a patient. Thepeptide can also include other amino terminal or carboxy terminal aminoacids. In some cases the additional amino acids protect the peptide,stabilize the peptide or alter the activity of the peptide. In somecases some or all of these additional amino acids are removed prior toadministration of the peptide to a patient. The peptide can include 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 80, 90, 100 or more aminoacids at its amino terminus or carboxy terminus or both. The number offlanking amino acids need not be the same. For example, there can be 10additional amino acids at the amino terminus of the peptide and none atthe carboxy terminus.

In certain embodiments the peptides include either one or two or morecontiguous negatively charged amino acids (e.g., Asp or Glu) or one ortwo or more contiguous positively charged residues (e.g., Lys or Arg) orone or two or more contiguous positively or negatively charged aminoacids at the carboxy terminus. In these embodiments all of the flankingamino acids at the carboxy terminus are either positively or negativelycharged. In other embodiments the carboxy terminal charged amino acidsare preceded by a Leu. For example, the following amino acid sequencescan be added to the carboxy terminus of the peptide: Asp; Asp Lys; LysLys Lys Lys Lys Lys (SEQ ID NO:93); Asp Lys Lys Lys Lys Lys Lys (SEQ IDNO:94); Leu Lys Lys; and Leu Asp. It is also possible to simply add Leuat the carboxy terminus.

In a first aspect, the invention features a polypeptide comprising,consisting of, or consisting essentially of the amino acid sequence:Xaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein:

-   -   Xaa₁ is Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is        missing;    -   Xaa₂ is His, Asp, Glu, Ala, Ser, Asn, Gly, or is missing;    -   Xaa₃ is Thr, Asp, Ser, Glu, Pro, Val or Leu;    -   Xaa₅ is Asp, Ile or Glu;    -   Xaa₆ is Ile, Trp or Leu;    -   Xaa₇ is Cys, Ser, or Tyr;    -   Xaa₈ is Ala, Val, Thr, Ile, Met or is missing;    -   Xaa₉ is a) any amino acid, b) Phe, Tyr, Asn, Trp, c) an amino        acid other than Phe, Trp, or Tyr, d) non-aromatic amino acid        or e) is missing;    -   Xaa₁₀ is Ala, Val, Met, Thr or Ile;    -   Xaa₁₁ is Ala or Val;    -   Xaa₁₃ is Ala or Thr;    -   Xaa₁₄ is Gly, Ala or Ser;    -   Xaa₁₅ is Cys, Tyr or is missing; and    -   Xaa₁₆ is: a) Trp, Tyr or Phe to create a chymotrypsin cleavage        site; b) Lys or Arg to create a trypsin cleavage site; c) is        missing or d) His or Leu or Ser.

In some embodiments, Xaa₁ is preceded by Lys or Tyr.

In certain embodiments, a Cys is replaces by any amino acid other thanCys. Certain such polypeptides will have fewer disulfide bonds.

In a related aspect the invention features a composition comprising apolypeptide comprising, consisting of, or consisting essentially of theamino acid sequence: Xaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein: Xaa₁ is Ser,Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is missing; Xaa₂ is His,Asp, Glu, Ala, Ser, Asn, Gly, Pro or is missing; Xaa₃ is Thr, Asp, Ser,Glu, Pro, Val or Leu; Xaa₅ is Asp, Ile or Glu; Xaa₆ is Ile, Trp or Leu;Xaa₇ is Cys, Ser, or Tyr; Xaa₈ is Ala, Val, Thr, Ile, Met or is missing;Xaa₉ is Phe, Tyr, Asn, Trp, an amino acid other than Phe, Trp, or Tyr,is a non-aromatic amino acid or is missing; Xaa₁₀ is Ala, Val, Met, Thror Ile; Xaa₁₁ is Ala or Val; Xaa₁₃ is Ala or Thr; Xaa₁₄ is Gly, Ala orSer; Xaa₁₅ is Cys, Tyr or is missing; and Xaa₁₆ is: a) Trp, Tyr or Pheto create a chymotrypsin cleavage site; b) Lys or Arg to create atrypsin cleavage site; c) is missing or d) His or Leu or Ser and apharmaceutically acceptable carrier. In related aspects, the inventionfeatures a pharmaceutically acceptable tablet, pill, capsule comprisingthe peptide.

In a related aspect, the invention features a polypeptide comprising,consisting of, or consisting essentially of the amino acid sequence:Xaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein:

-   -   Xaa₁ is Asn, any amino acid or is missing;    -   Xaa₂ is Asp, Glu, any amino acid or is missing;    -   Xaa₃ is Asp or Glu;    -   Xaa₅ is any amino acid or Glu;    -   Xaa₆ is any amino acid or Leu;    -   Xaa₇ is Cys;    -   Xaa₈ is any amino acid or Val;    -   Xaa₉ is Asn, Gln, Tyr;    -   Xaa₁₀ is is any amino acid or Val;    -   Xaa₁₁ is any amino acid or Ala;    -   Xaa₁₃ is is any amino acid or Thr;    -   Xaa₁₄ is is any amino acid or Gly;    -   Xaa₁₅ is Cys;    -   Xaa₁₆ is any amino acid, Leu or missing

In a related aspect, the invention features a polypeptide comprising,consisting of, or consisting essentially of the amino acid sequence:Asn, Xaa₂ Xaa₃ Xaa₄ Glu₅ Leu₆ Xaa₇ Val₈ Asn₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Thr₁₃Xaa₁₄ Xaa₁₅ Leu₁₆ (SEQ ID NO: 2)

-   -   Xaa₂ is Asp or Glu;    -   Xaa₃ is Asp or Glu;    -   Xaa₄ is Cys or Mpt (mercaptoproline) or Pen (penicillamine) or        Dpr (diaminopropionic acid) or Asp or Glu;    -   Xaa₇ is Cys or Mpt (mercaptoproline) or Pen (penicillamine) or        Dpr (diaminopropionic acid) or Asp or Glu;    -   Xaa₁₀ is Val or Pro;    -   Xaa₁₁ is Ala or Aib (alpha-aminoisobutyric acid);    -   Xaa₁₂ is Cys or Mpt (mercaptoproline) or Pen (penicillamine) or        Dpr (diaminopropionic acid) or Asp or Glu;    -   Xaa₁₄ is Gly or Ala;    -   Xaa₁₅ is Cys or Mpt (mercaptoproline) or Pen (penicillamine) or        Dpr (diaminopropionic acid) or Asp or Glu; and

In certain embodiments, where Xaa₁₅ is other than Cys or is missing,Xaa₇ is Ser or an amino acid other than Cys.

In certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of Xaa₁,Xaa₂, Xaa₃, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₃, Xaa₁₄,and Xaa₁₆ are any amino acid other than Cys.

In certain embodiments, Xaa₉ is any amino acid other than Gln. In otherembodiments where Xaa₂ and Xaa₃ are Glu, Xaa₉ is any amino acid otherthan Gln.

In certain embodiments Xaa₁ and Xaa₂ are missing; Xaa₃ is Thr; Xaa₅ isGlu; Xaa₆ is Ile or Leu; Xaa₈ is Ala, Val, or Ile; Xaa₉ is Phe or Tyr;Xaa₁₀ is Ala or Val; Xaa₁₁ is Ala; Xaa₁₃ is Ala or Thr; Xaa₁₄ is Gly;and Xaa₁₆ is Trp, Tyr, Phe, Lys, Arg or is missing.

In certain embodiments the polypeptide comprising, consisting of, orconsisting essentially of the amino acid sequence: Xaa₁ Xaa₂ Xaa₃ Cys₄Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQID NO:1) is not cleaved after Xaa₉ by chymotrypsin. In these embodimentswherein:

-   -   Xaa₁ is Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is        missing;    -   Xaa₂ is His, Asp, Glu, Ala, Ser, Asn, or Gly, or is missing;    -   Xaa₃ is Thr, Asp, Ser, Glu, Pro, Val or Leu or is missing;    -   Xaa₅ is Asp, Ile or Glu;    -   Xaa₆ is Ile, Trp or Leu;    -   Xaa₇ is Cys, Ser, or Tyr;    -   Xaa₈ is Ala, Val, Thr, Ile, Met or is missing;    -   Xaa₉ is either: a) any amino acid other than Phe and Tyr, b) any        amino acid other than Phe, Tyr, and Trp, c) any amino acid other        than Phe, Tyr, Trp, Ile, Leu and Val; d) any amino acid other        than Phe, Tyr, Trp, Ile, Leu, Val, and His; d) any non-aromatic        amino acid or e) is missing;    -   Xaa₁₀ is Ala, Val, Met, Thr or Ile;    -   Xaa₁₁ is Ala or Val;    -   Xaa₁₃ is Ala or Thr;    -   Xaa₁₄ is Gly, Ala or Ser;    -   Xaa₁₅ is Cys, Tyr or is missing; and    -   Xaa₁₆ is: a) Trp, Tyr or Phe to create a chymotrypsin cleavage        site; b) Lys or Arg to create a trypsin cleavage site; c) is        missing or d) His or Leu or Ser.

In addition, the invention features variants of Xaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ IDNO:1) that is not cleaved after Xaa₉ by chymotrypsin due to the additionof an amino terminal lysine. An example of such a molecule is a humanguanylin variant having an amino terminal lysine: KPGTCEICAYAACTGC (SEQID NO: 3).

In certain embodiments of the peptide comprising, consisting of, orconsisting essentially of the amino acid sequence: Xaa₁ Xaa₂ Xaa₃ Cys₄Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQID NO:1) that is not cleaved after Xaa₉ by chymotrypsin, Xaa₇ and Xaa₁₅are both Cys.

Also within the invention are variants of PGTCEICAYAACTGC (humanguanylin) (SEQ ID NO: 4) wherein Y is substituted by any amino acidother than a) Phe; b) any amino acid other than Phe and Trp; c) anyamino acid other than Phe, Trp, Ile, Leu and Val; d) any amino acidother than Phe, Trp, Ile, Leu, Val and His; e) any non-aromatic aminoacid or f) is missing.

In certain embodiments the polypeptide comprising, consisting of, orconsisting essentially of the amino acid sequence: Xaa₁ Xaa₂ Xaa₃ Cys₄Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQID NO:1) is not cleaved after Xaa₉ by either chymotrypsin or trypsin. Inthese embodiments wherein:

-   -   Xaa₁ is Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or is        missing;    -   Xaa₂ is His, Asp, Glu, Ala, Ser, Asn, or Gly, or is missing;    -   Xaa₃ is Thr, Asp, Ser, Glu, Pro, Val or Leu or is missing;    -   Xaa₅ is Asp, Ile or Glu;    -   Xaa₆ is Ile, Trp or Leu;    -   Xaa₇ is Cys, Ser, or Tyr;    -   Xaa₈ is Ala, Val, Thr, Ile, Met or is missing;    -   Xaa₉ is either: a) any amino acid other than Lys, Arg, Phe and        Tyr, b) any amino acid other than Lys, Arg, Phe, Tyr, and        Trp, c) any amino acid other than Lys, Arg, Phe, Tyr, Trp, Ile,        Leu and Val; d) any amino acid other than Lys, Arg, Phe, Tyr,        Trp, Ile, Leu, Val, and His; or e) is missing;    -   Xaa₁₀ is Ala, Val, Met, Thr or Ile;    -   Xaa₁₁ is Ala or Val;    -   Xaa₁₃ is Ala or Thr;    -   Xaa₁₄ is Gly, Ala or Ser;    -   Xaa₁₅ is Cys, Tyr or is missing; and    -   Xaa₁₆ is: a) Trp, Tyr or Phe to create a chymotrypsin cleavage        site; b) Lys or Arg to create a trypsin cleavage site; c) is        missing or d) His or Leu or Ser.

In certain embodiments of the peptide comprising, consisting of, orconsisting essentially of the amino acid sequence: Xaa₁ Xaa₂ Xaa₃ Cys₄Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQID NO:1) that is not cleaved after Xaa₉ by chymotrypsin or trypsin, Xaa₇and Xaa₁₅ are both Cys.

Useful variants of PGTCEICAYAACTGC (human guanylin) (SEQ ID NO: 4) thatshould not be cleaved by chymotrypsin include:

PGTCEICASAACTGC (SEQ ID NO: 5) PGTCEICATAACTGC (SEQ ID NO: 6)PGTCEICANAACTGC (SEQ ID NO: 7) PGTCEICAQAACTGC (SEQ ID NO: 8)PGTCEICARAACTGC (SEQ ID NO: 9) PGTCEICAEAACTGC (SEQ ID NO: 10)PGTCEICADAACTGC (SEQ ID NO: 11) PGTCEICAGAACTGC (SEQ ID NO: 12)PGTCEICAAAACTGC (SEQ ID NO: 13) PGTCEICAMAACTGC. (SEQ ID NO: 14)

Additional variants which are not likely to be cleaved by chymotrypsinunder certain conditions include:

PGTCEICAIAACTGC (SEQ ID NO: 15) PGTCEICALAACTGC (SEQ ID NO: 16)PGTCEICAVAACTGC (SEQ ID NO: 17) PGTCEICAHAACTGC (SEQ ID NO: 18)

The invention also features deletion variants of any of the peptidesdescribed herein in which one, two, three or four amino acids, otherthan a Cys, are deleted. Where two (or more) amino acids are deleted andthe peptide comprises the sequence: Cys_(a) Xaa Xaa Cys_(b) Xaa Xaa XaaXaa Cys_(c) Xaa Xaa Cys_(d), in some embodiments two or more deletionscan be located between Cys_(a) and Cys_(b) or between Cys_(b) andCys_(c) or between Cys_(c) and Cys_(d). Thus, there can be two or moredeletions between two Cys. However, in other embodiments there is atmost one deletion between each Cys, i.e., there is no more than onedeletion between each of Cys_(a) and Cys_(b), Cys_(b) and Cys_(c), andCys_(c) and Cys_(d). Thus, the invention includes any of the peptidesdescribed herein comprising the sequence Cys_(a) Xaa Xaa Cys_(b) Xaa XaaXaa Xaa Cys_(c) Xaa Xaa Cys_(d) wherein: a) one amino acid betweenCys_(a) and Cys_(b) is deleted; b) one amino acid between Cys_(b) andCys_(c) is deleted; c) one amino acid between Cys_(c) and Cys_(d) isdeleted; d) one amino acid between Cys_(a) and Cys_(b) is deleted andone amino acid between Cys_(b) and Cys_(c) is deleted; e) one amino acidbetween Cys_(a) and Cys_(b) is deleted and one amino acid between Cys,and Cys_(d) is deleted; f) one amino acid between Cys_(b) and Cys, isdeleted and one amino acid between Cys_(c) and Cys_(d) is deleted; or g)one amino acid between Cys_(a) and Cys_(b) is deleted, one amino acidbetween Cys_(b) and Cys_(c) is deleted, and one amino acid between Cys,and Cys_(d) is deleted. In addition, one or more amino acids precedingCys_(a) and/or one or more amino acids following Cys_(d) can be deleted.The various deletion variants are peptides that bind to and/or activatethe GC-C receptor.

The invention also features deletion variants of any of the peptidesdescribed herein in which one, two, three or four amino acids (ornon-natural amino acids or natural or non-natural amino acid analogs),other than a Cys (or an amino acid substituted for Cys, e.g., an aminoacid capable of forming a covalent bond to another amino acid) isdeleted. Thus, additional variants include those in which a Cys issubstituted by an amino acid capable of forming a covalent linkage withanother amino acid (e.g., a Cys or a substitute therefore). Such aminoacids include: Mpt (mercaptoproline) or Pen (penicillamine) or Dpr(diaminopropionic acid).

FIG. 1 includes deletion variants of human guanylin in which one, two,three or four amino acids are deleted. The deleted amino acids arebetween Cys_(a) and Cys_(d) as well as amino terminal to Cys_(a).

The invention also features insertion variants of any of the peptidesdescribed herein in which one, two, three or four amino acids areinserted.

Where two (or more) amino acids are inserted and the peptide comprisesthe sequence: Cys_(a) Xaa Xaa Cys_(b) Xaa Xaa Xaa Xaa Cys_(c) Xaa XaaCys_(d), in some embodiments two or more insertions can be locatedbetween Cys_(a) and Cys_(b) or between Cys_(b) and Cys_(c) or betweenCys_(c) and Cys_(d). However, in other embodiments there is at most oneinsertion between each of Cys_(a) and Cys_(b) or between Cys_(b) andCys, or between Cys_(c) and Cys_(d). Thus, the invention includes any ofthe peptides described herein comprising the sequence Cys_(a) Xaa XaaCys_(b) Xaa Xaa Xaa Xaa Cys_(c) Xaa Xaa Cys_(d) wherein: a) one aminoacid is inserted between Cys_(a) and Cys_(b); b) one amino acid isinserted between Cys_(b) and Cys_(c); c) one amino acid is insertedbetween Cys, and Cys_(d); d) one amino acid is inserted between Cys_(a)and Cys_(b) and one amino acid is inserted between Cys_(b) and Cys_(c);e) one amino acid is inserted between Cys_(a) and Cys_(b) and one aminoacid is inserted between Cys_(c) and Cys_(d); f) one amino acid isinserted between Cys_(c), and Cys, and one amino acid is insertedbetween Cys_(c) and Cys_(d) or g) one amino acid is inserted betweenCys_(a) and Cys_(b), one amino acid is inserted between Cys_(b) andCys_(c), and one amino acid is inserted between Cys, and Cys_(d). Inaddition, one or more amino acids can be inserted preceding Cys_(a)and/or one or more amino acids can be inserted following Cys_(d). Theinsertions can be any natural or non-natural occurring amino acid (e.g.,Gly or Ala) or amino acid analog and where there are more than oneinsertions present, they can be the same or different. The variousdeletion variants are peptides that bind to and/or activate the GC-Creceptor.

For example, the invention includes the following insertion variants ofPGTCGEICAYAACTGC (human guanylin) (SEQ ID NO:19) include:

PGTCEGICAYAACTGC (SEQ ID NO: 20) PGTCEIGCAYAACTGC (SEQ ID NO: 21)PGTCEICGAYAACTGC (SEQ ID NO: 22) PGTCEICAGYAACTGC (SEQ ID NO: 23)PGTCEICAYGAACTGC (SEQ ID NO: 24) PGTCEICAYAGACTGC (SEQ ID NO: 25)PGTCEICAYAAGCTGC (SEQ ID NO: 26) PGTCEICAYAACGTGC (SEQ ID NO: 27)PGTCEICAYAACTGGC (SEQ ID NO: 28) PGTCAEICAYAACTGC (SEQ ID NO: 29)PGTCEAICAYAACTGC (SEQ ID NO: 30) PGTCEIACAYAACTGC (SEQ ID NO: 31)PGTCEICAAYAACTGC (SEQ ID NO: 32) PGTCEICAYAAACTGC (SEQ ID NO: 33)PGTCEICAYAACATGC (SEQ ID NO: 34) PGTCEICAYAACTAGC (SEQ ID NO: 35)PGTCEICAYAACTGAC (SEQ ID NO: 36) PGTCAEICAAYAACTGC (SEQ ID NO: 37)PGTCEAICAAYAACTGC (SEQ ID NO: 38) PGTCEIACAAYAACTGC (SEQ ID NO: 39)

Other insertion variants of human guanylin can have up to four aminoacids (i.e., 0, 1, 2, 3 or 4 natural or non-natural amino acids)inserted after each of the 15 amino acids in human guanylin. Thus, theinvention includes peptides having the sequence: Pro Xaa₍₀₋₄₎ GlyXaa₍₀₋₄₎ Thr Xaa₍₀₋₄₎ Cys Xaa₍₀₋₄₎ Glu Xaa₍₀₋₄₎ Ile Xaa₍₀₋₄₎ CysXaa₍₀₋₄₎ Ala Xaa₍₀₋₄₎ Tyr Xaa₍₀₋₄₎ Ala Xaa₍₀₋₄₎ Ala Xaa₍₀₋₄₎ CysXaa₍₀₋₄₎ Thr Xaa₍₀₋₄₎ Gly Xaa₍₀₋₄₎ Cys Xaa₍₀₋₄₎ (SEQ ID NO: 95). Theinserted amino acids can be any amino acid and can be the same ordifferent. In certain embodiments the inserted amino acids are all Glyor all Ala or a combination of Gly and Ala.

FIG. 2 depicts insertion variants of human guanylin in which one, two,three or four amino acids are inserted. The inserted amino acids arebetween Cys_(a) and Cys_(d) as well as amino terminal to Cys_(a) andcarboxy terminal to Cys_(d).

The invention also features variants of peptides having the sequenceXaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ ID NO:1), e.g., variants of PGTCEICAYAACTGC humanguanylin (SEQ ID NO: 4) in which up to four amino acids are deletedand/or up to four amino acids are inserted. The insertions and deletionscan be between Cys4 and Cys12 in SEQ ID NO:1 or they can be aminoterminal to Cys4 and/or carboxy terminal to Cys12 in SEQ ID NO:1

When Xaa₁₆ is Trp, Tyr or Phe, the peptide has a chymotrypsin cleavagesite that is located at a position where cleavage will liberate theportion of the peptide carboxy-terminal to Xaa₁₆. When Xaa₁₆ is Lys orArg, the peptide has a trypsin cleavage site that is located at aposition where cleavage will liberate portion of the peptidecarboxy-terminal to Xaa₁₆. Thus, if the peptide includes an analgesicpeptide carboxy-terminal to Xaa₁₆, the peptide will be liberated in thedigestive tract upon exposure to the appropriate protease. Among theanalgesic peptides which can be included in the peptide are: AspPhe,endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, andsubstance P and other analgesic peptides described herein.

When Xaa₁ or the amino-terminal amino acid of the peptide of theinvention (e.g., Xaa₂ or Xaa₃) is Trp, Tyr or Phe, the peptide has achymotrypsin cleavage site that is located at a position where cleavagewill liberate the portion of the peptide amino-terminal to Xaa₁ (or Xaa₂or Xaa₃) along with Xaa₁, Xaa₂ or Xaa₃. When Xaa₁ or the amino-terminalamino acid of the peptide of the invention (e.g., Xaa₂ or Xaa₃) is Lysor Arg, the peptide has a trypsin cleavage site that is located at aposition where cleavage will liberate portion of the peptideamino-terminal to Xaa₁ along with Xaa₁, Xaa₂ or Xaa₃). Thus, forexample, if the peptide includes an analgesic peptide amino-terminal toXaa₁, the peptide will be liberated in the digestive tract upon exposureto the appropriate protease. Among the analgesic peptides which can beincluded in the peptide are: AspPhe, endomorphin-1, endomorphin-2,nocistatin, dalargin, lupron, and substance p and other analgesicpeptides described herein.

The peptides can linked, e.g., covalently linked to any of a variety ofother analgesic peptides or analgesic compounds. Thus, a peptidedescribed herein can be linked to a second therapeutic agent, e.g., anagent for treating constipation (e.g., a chloride channel activator suchas SPI-0211; Sucampo Pharmaceuticals, Inc.; Bethesda, Md., a laxativesuch as MiraLax; Braintree Laboratories, Braintree Mass.) or some othergastrointestinal disorder. Examples of a second therapeutic agentinclude: acid reducing agents such as proton pump inhibitors (e.g.,omeprazole, esomeprazole, lansoprazole, pantorazole and rabeprazole), H2receptor blockers (e.g., cimetidine, ranitidine, famotidine andnizatidine), pro-motility agents such as motilin agonists (e.g., GM-611or mitemcinal fumarate), 5HT receptor agonists (e.g., 5HT4 receptoragonists such as Zelnorm®; 5HT3 receptor agonists such as MKC-733), 5HTreceptor antagonists (e.g., 5HT1, 5HT2, 5HT3 (e.g., alosetron), 5HT4receptor antagonists, muscarinic receptor agonists, anti-inflammatoryagents, antispasmodics, antidepressants, centrally-acting analgesicagents such as opioid receptor agonists, opioid receptor antagonists(e.g., naltrexone), agents for the treatment of Inflammatory boweldisease, Crohn's disease and ulcerative colitis (e.g., Traficet-EN™(ChemoCentryx, Inc.; San Carlos, Calif.), agents that treatgastrointestinal or visceral pain, and cGMP phosphodiesterase inhibitors(motapizone, zaprinast, and suldinac sulfone). The peptides of theinvention can also be linked to agents such a tianeptine (Stablon®) andother agents described in U.S. Pat. No. 6,683,072; (E)-4(1,3bis(cyclohexylmethyl)-1,2,3,4,-tetrahydro-2,6-diono-9H-purin-8-yl)cinnamicacid nonaethylene glycol methyl ether ester and related compoundsdescribed in WO 02/067942. The peptides can be linked to an agentselected from the group consisting of: Ca channel blockers (e.g.,ziconotide), complete or partial 5HT receptor antagonists (for example5HT3 (e.g., alosetron, ATI-7000; Aryx Thearpeutics, Santa Clara Calif.),5HT4, 5HT2, and 5HT1 receptor antagonists), complete or partial 5HTreceptor agonists including 5HT3, 5HT2, 5HT4 (e.g., tegaserod, mosaprideand renzapride) and 5HT1 receptor agonists, CRF receptor agonists(NBI-34041), β-3 adrenoreceptor agonists, opioid receptor agonists(e.g., loperamide, fedotozine, and fentanyl, naloxone, naltrexone,methyl nalozone, nalmefene, cypridime, beta funaltrexamine,naloxonazine, naltrindole, and nor-binaltorphimine, morphine,diphenyloxylate, enkephalin pentapeptide, asimadoline, and trimebutine),NK1 receptor antagonists (e.g., ezlopitant and SR-14033), CCK receptoragonists (e.g., loxiglumide), NK1 receptor antagonists, NK3 receptorantagonists (e.g., talnetant, osanetant (SR-142801), SSR-241586),norepinephrine-serotonin reuptake inhibitors (NSRI; e.g., milnacipran),vanilloid and cannabanoid receptor agonists (e.g., arvanil), sialorphin,sialorphin-related peptides comprising the amino acid sequence QHNPR(SEQ ID NO: 85) for example, VQHNPR (SEQ ID NO: 86); VRQHNPR (SEQ ID NO:87); VRGQHNPR (SEQ ID NO: 88); VRGPQHNPR (SEQ ID NO: 89); VRGPRQHNPR(SEQ ID NO: 90); VRGPRRQHNPR (SEQ ID NO: 91); and RQHNPR (SEQ ID NO:92), compounds or peptides that are inhibitors of neprilysin,frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1), loperamide,Tyr-Arg (kyotorphin), CCK receptor agonists (caerulein), conotoxinpeptides, pepetide analogs of thymulin, loxiglumide, dexloxiglumide (theR-isomer of loxiglumide) (WO 88/05774) and other analgesic peptides orcompounds.

Amino acid, non-amino acid, peptide and non-peptide spacers can beinterposed between a peptides of the invention and a peptide that hassome other biological function, e.g., an analgesic peptide or a peptideused to treat obesity. The linker can be one that is cleaved from theflanking peptides in vivo or one that remains linked to the flankingpeptides in vivo. For example, glycine, beta-alanine, glycyl-glycine,glycyl-beta-alanine, gamma-aminobutyric acid, 6-aminocaproic acid,L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine can beused as a spacer (Chaltin et al. 2003 Helvetica Chimica Acta 86:533-547;Caliceti et al. 1993 FARMCO 48:919-32) as can polyethylene glycols(Butterworth et al. 1987 J. Med. Chem 30:1295-302) and maleimidederivatives (King et al. 2002 Tetrahedron Lett. 43:1987-1990). Variousother linkers are described in the literature (Nestler 1996 MolecularDiversity 2:35-42; Finn et al. 1984 Biochemistry 23:2554-8; Cook et al.1994 Tetrahedron Lett. 35:6777-80; Brokx et al. 2002 Journal ofControlled Release 78:115-123; Griffin et al. 2003 J. Am. Chem. Soc.125:6517-6531; Robinson et al. 1998 Proc. Natl. Acad. Sci. USA95:5929-5934.

The peptides can include the amino acid sequence of a peptide thatoccurs naturally in a vertebrate (e.g., mammalian) species or in abacterial species. In addition, the peptides can be partially orcompletely non-naturally occurring peptides. Also within the inventionare peptidomimetics corresponding to the peptides of the invention.

When fully folded, disulfide bonds are present between the first andthird cysteines and between the second and fourth cysteines, e.g., thereis a disulfide bond between Cys₄ and Cys₁₂ and a disulfide bond betweenXaa₇ and Xaa₁₅ (when Xaa₇ is a Cys and Xaa₁₅ is a Cys). In someembodiments, the peptide has only one disulfide bond, e.g., between thefirst and third cysteines (i.e., Cys₄ and Cys₁₂; corresponds to thefirst and second cysteines when Xaa₇ is other than Cys). In certainembodiments one or more Cys can be replaced by Mpt (mercaptoproline) orPen (penicillamine) or Dpr (diaminopropionic acid) or some other aminoacid that can covalently link to another amino acid (e.g., Cys, Mpt, Penor Dpr). In some embodiments, one or both members of a pair of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, 3-mercaptoproline (Kolodziej et al. 1996 Int J PeptProtein Res 48:274); 4, a dimethylcysteine (Hunt et al. 1993 Int J PeptProtein Res 42:249) or diaminopropionic acid (Smith et al. 1978 J MedChem 21:117) to form alternative internal cross-links at the positionsof the normal disulfide bonds.

In addition, one or more disulfide bonds can be replaced by alternativecovalent cross-links, e.g., an amide bond, an ester linkage, an alkyllinkage, a thio ester linkage, a lactam bridge, a carbamoyl linkage, aurea linkage, a thiourea linkage, a phosphonate ester linkage, an alkyllinkage, and alkenyl linkage, an ether, a thioether linkage, or an aminolinkage. For example, Ledu et al. (Proceedings Nat'l Acad. Sci.100:11263-78, 2003) described methods for preparing lactam and amidecross-links. Schafmeister et al. (J. Am. Chem. Soc. 122:5891, 2000)describes stable, all carbon cross-links. In some cases, the generationof such alternative cross-links requires replacing the Cys residues withother residues such as Lys or Glu or non-naturally occurring aminoacids.

In certain embodiments one or more amino acids can be replaced by anon-naturally occurring amino acid or a naturally or non-naturallyoccurring amino acid analog. For example, an aromatic amino acid can bereplaced by 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine,triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine(norTyr). Phg and norTyr and other amino acids including Phe and Tyr canbe substituted by, e.g., a halogen, —CH3, —OH, —CH₂NH₃, —C(O)H, —CH₂CH₃,—CN, —CH₂CH₂CH₃, —SH, or another group.

Further examples of unnatural amino acids include: an unnatural analogueof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α,α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; ap-acetyl-L-phenylalanine; an 0-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAcβ-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; aisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine; an amino-,isopropyl-, or O-allyl-containing phenylalanine analogue; a dopa,O-methyl-L-tyrosine; a glycosylated amino acid; ap-(propargyloxy)phenylalanine, dimethyl-Lysine, hydroxy-proline,mercaptopropionic acid, methyl-lysine, 3-nitro-tyrosine, norleucine,pyro-glutamic acid, Z (Carbobenzoxyl), c-Acetyl-Lysine, 3-alanine,aminobenzoyl derivative, aminobutyric acid (Abu), citrulline,aminohexanoic acid, aminoisobutyric acid, cyclohexylalanine,d-cyclohexylalanine, hydroxyproline, nitro-arginine,nitro-phenylalanine, nitro-tyrosine, norvaline, octahydroindolecarboxylate, ornithine, penicillamine, tetrahydroisoquinoline,acetamidomethyl protected amino acids and a pegylated amino acid.Further examples of unnatural amino acids can be found in U.S.20030108885, U.S. 20030082575, and the references cited therein.

In some embodiments, an amino acid can be replaced by anaturally-occurring, non-essential amino acid, e.g., taurine.

Methods to manufacture peptides containing unnatural amino acids can befound in, for example, U.S. 20030108885, U.S. 20030082575, Deiters etal., J Am Chem Soc. (2003) 125:11782-3, Chin et al., Science (2003)301:964-7, and the references cited therein.

Peptides that include non-natural amino acids can also be prepared usingthe methods described in WO02086075.

The peptides of the invention can be modified using standardmodifications. Modifications may occur at the amino (N-), carboxy (C-)terminus, internally or a combination of any of the preceeding. In oneaspect of the invention, there may be more than one type of modificationon the peptide. Modifications include but are not limited to:acetylation, amidation, biotinylation, cinnamoylation, farnesylation,formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyror Thr), stearoylation, succinylation, sulfurylation and cyclisation(via disulfide bridges or amide cyclisation), and modification by Cy3 orCy5. The peptides of the invention may also be modified by2,4-dinitrophenyl (DNP), DNP-lysin, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine). The peptidesof the invention may also be conjugated to, for example, BSA or KLH(Keyhole Limpet Hemocyanin).

The invention also features a purified polypeptide comprising,consisting of or consisting essentially of the amino acid sequence: Xaa₁Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein:

-   -   Xaa₁ is any amino acid or is missing;    -   Xaa₂ is any amino acid or is missing;    -   Xaa₃ is any amino acid or is missing;    -   Xaa₅ is Glu;    -   Xaa₆ is Tyr, Trp, Phe or Leu;    -   Xaa₇ is Cys;    -   Xaa₈ is any of the 20 naturally-occurring amino acids other than        Cys or is missing;    -   Xaa₉ is any of the 20 naturally-occurring amino acids;    -   Xaa₁₀ is Pro or Gly;    -   Xaa₁₁ is any of the 20 naturally-occurring amino acids;    -   Xaa₁₃ is Thr, Val or Gly;    -   Xaa₁₄ is Gly or Ala;    -   Xaa₁₅ is Cys; and    -   Xaa₁₆ is any of the 20 naturally-occurring amino acids or is        missing.

In various embodiments: Xaa₉ is Asn; Xaa₁₁ is Ala or Thr; Xaa₈ ismissing; and Xaa₁₆ is Tyr.

In other embodiments Xaa4 is immediately preceded by an amino acidsequence selected from: Ser His Thr; Pro Ser Thr; Thr; Pro Asp Pro; IleAla Glu Asp Ser His Thr (SEQ ID NO:8604); Ile Ala Gln Asp Pro Ser Thr(SEQ ID NO:8605); Ala Asn Thr; Asn Thr; Asp Pro Asn Thr (SEQ IDNO:8606); Lys Asn Thr; Pro Asn Thr; Ile Ala Gln Asp Pro Asn Thr (SEQ IDNO:8607); Lys Pro Asn Thr (SEQ ID NO:8608); Asp Pro Gly Thr (SEQ IDNO:8609); Glu Asp Pro Gly Thr (SEQ ID NO:8610); Pro Gly Thr; Pro AlaThr; Val Ala Ala Arg Ala Asp Leu (SEQ ID NO:8611); Gly Asp Asp; Asn AspGlu; Gln Glu Asp; Asn Asp Asp; Arg Thr Ile Ala Asn Asp Asp (SEQ IDNO:8612); Thr Ile Ala Asn Asp Asp (SEQ ID NO:8613); Asp Asp; Arg Thr MetAsp Asn Asp Glu (SEQ ID NO:8614); Arg Thr Ile Ala Gly Asp Asp (SEQ IDNO:8615); Arg Thr Ile Ala Asn Asp (SEQ ID NO:8616); Asp; Glu Asp; ArgSer Ile Ser Gin Glu Asp (SEQ ID NO:8617); Thr Asp Glu; Arg Thr Ile AlaThr Asp Glu (SEQ ID NO:8618); Glu; Ile Ile Thr Pro Pro Asp Pro (SEQ IDNO:8619); Gln Glu Leu; Lys Asp Asp; Gln Glu Glu; Arg Tyr Ile Asn Gln GluGlu (SEQ ID NO:8620); Ala Ser Ser Tyr Ala Ser (SEQ ID NO:8621); and ThrSer Ser Tyr Ala Ser (SEQ ID NO:8622).

The invention further features a purified polypeptide comprising,consisting of or consisting essentially the amino acid sequence: Xaa₁Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂ Xaa₁₃ Xaa₁₄Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein:

-   -   Xaa₁ is: a) Ser, Asn, Tyr, Ala, Gln, Pro, Lys, Gly, or Thr, or        is missing; b) preceded by Lys or Tyr; c) any amino acid; d)        missing; e) any amino acid other than Cys; or f) Lys or Arg;    -   Xaa₂ is: a) His, Asp, Glu, Ala, Ser, Asn, Gly, or is missing; b)        His, Asp, Glu, Ala, Ser, Asn, Gly, Pro or is missing; c) Asp,        Glu, any amino acid or is missing; d) Asp or Glu; e) any amino        acid other than Cys; e) Glu; f) missing; g) Trp, Tyr or Phe;        or h) Lys or Arg;    -   Xaa₃ is: a) Thr, Asp, Ser, Glu, Pro, Val or Leu; Asp or Glu; b)        any amino acid other than Cys; c) Glu; d) Thr; e) Thr, Asp, Ser,        Glu, Pro, Val or Leu or is missing; f) Tip, Tyr or Phe; or g)        Lys or Arg;    -   Xaa₄ is: a) Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr        (diaminopropionic acid), Asp, or Glu;    -   Xaa₅ is: a) any amino acid; b) Glu, Asp, Gln, Gly or Pro; c)        Glu; d) Glu or Asp; e) Asp, Ile or Glu; f) any amino acid; or g)        any amino acid other than Cys;    -   Xaa₆ is: a) Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; b)        Leu, Ile, Val, Lys, Arg, Trp, Tyr or Phe; Leu, Ile, Lys, Arg,        Trp, Tyr or Phe; c) Leu, Ile, Val, Trp, Tyr or Phe; d) Trp, Tyr,        Phe or Leu; e) Leu, Ile or Val; f) Ile, Trp or Leu; g) Trp, Tyr        or Phe; h) Ile or Leu; i) Tyr; j) any amino acid; k) any amino        acid except Leu; l) any natural or non-natural aromatic amino        acid; or m) any amino acid other than Cys;    -   Xaa₇ is: a) Cys, Ser, or Tyr; Cys; b) Cys, Mpt        (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic        acid), Asp or Glu; c) Ser; or d) an amino acid other than Cys;    -   Xaa₈ is: a) Ala, Val, or Ile; b) Ala, Val, Thr, Ile, Met or is        missing; c) any amino acid; d) Val; e) any amino acid other than        Cys; or f) missing;    -   Xaa₉ is: a) any amino acid; b) any amino acid other than Phe and        Tyr; c) any amino acid other than Phe, Tyr, and Trp; d) any        amino acid other than Phe, Tyr, Trp, Ile, Leu and Val; e) any        amino acid other than Phe, Tyr, Trp, Ile, Leu, Val, and His; f)        any amino acid other than Gln; g) any amino acid other than Lys,        Arg, Phe, Tyr, and Trp; h) any amino acid other than Lys, Arg,        Phe, Tyr, Trp, Ile, Leu and Val; i) any amino acid other than        Lys, Arg, Phe, Tyr, Trp, Ile, Leu, Val, and His; j) any        non-aromatic amino acid; k) missing; l) Phe, Tyr, Asn, or        Trp; m) Asn, Tyr, Asp or Ala; n) Asn, Gln, or Tyr; o) Phe or        Tyr; p) Asn; or q) any amino acid other than Cys;    -   Xaa₁₀ is: a) Ala, Pro or Gly; b) Pro or Gly; c) Pro; d) Ala,        Val, Met, Thr or Ile; e) any amino acid; f) Val; g) Val or        Pro; h) Ala or Val; i) any amino acid other than Cys; j) Pro;        or k) Gly;    -   Xaa₁₁ is: a) any amino acid; b) Ala, Leu, Ser, Gly, Val, Glu,        Gln, Ile, Leu, Lys, Arg, or Asp; c) Ala or Gly; d) Ala; e) Ala        or Val; f) any amino acid; g) Ala or Aib (alpha-aminoisobutyric        acid); h) any amino acid other than Cys; i) Ala or Thr; or j)        Thr.    -   Xaa₁₂ is: a) Cys, Mpt (mercaptoproline), Pen (penicillamine),        Dpr (diaminopropionic acid), Asp, or Glu; or b) any amino acid        other than Cys;    -   Xaa₁₃ is: a) Thr, Ala, Asn, Lys, Arg, or Trp; b) Thr, Ala, Lys,        Arg, or Trp; c) any amino acid; d) any non-aromatic amino        acid; e) Thr, Ala, or Trp; f) Trp, Tyr or Phe; g) Thr or Ala; h)        any amino acid; i) Thr; j) any amino acid other than Cys; k)        Thr, Val, or Gly; 1) Thr or Val, m) Thr or Gly, n) Val or        Thr; o) Val; p) Thr; or q) Gly;    -   Xaa₁₄ is: a) Gly, Pro or Ala; b) Gly; c) any amino acid; d) Gly,        Ala or Ser; e) Gly or Ala; f) any amino acid other than Cys;        or g) Ala;    -   Xaa₁₅ is: a) Cys, Tyr or is missing; b) Cys; c) Cys, Mpt        (mercaptoproline), Pen (penicillamine), Dpr (diaminopropionic        acid), Asp, Glu; or d) any amino acid other than Cys or is        missing; and    -   Xaa₁₆ is: a) Trp, Tyr, Phe, Asn, Ile, Val, His or Leu; b) Trp,        Tyr, Phe, Asn or Leu; c) Trp, Tyr, Phe or Leu; d) Trp, Tyr, or        Phe; e) Leu, Ile or Val; f) His, Leu or Ser; g) Tyr or Leu; Lys        or Arg; h) His; i) any amino acid, j) Leu, or missing; k) Trp,        Tyr, Phe, Lys, Arg or is missing; l) missing; m) any amino acid        other than Cys; or n) Tyr.

Also featured is purified polypeptide comprising, consisting of orconsisting essentially of the amino acid sequence: Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQID NO:1) wherein:

-   -   Xaa₁ is any amino acid or is missing;    -   Xaa₂ is any amino acid or is missing;    -   Xaa₃ is any amino acid or is missing;    -   Xaa₄ is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr        (diaminopropionic acid), Asp or Glu;    -   Xaa₅ is Glu;    -   Xaa₆ is Tyr, Trp, Phe or Leu;    -   Xaa₇ is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr        (diaminopropionic acid), Asp or Glu;    -   Xaa₈ is any amino acid other than Cys or is missing;    -   Xaa₉ is any amino acid;    -   Xaa₁₀ is Pro or Gly;    -   Xaa₁₁ is any amino acid;    -   Xaa₁₂ is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr        (diaminopropionic acid), Asp or Glu;    -   Xaa₁₃ is Thr, Val or Gly;    -   Xaa₁₄ is Gly or Ala;    -   Xaa₁₅ is Cys, Mpt (mercaptoproline), Pen (penicillamine), Dpr        (diaminopropionic acid), Asp or Glu; and    -   Xaa₁₆ is any amino acid or is missing.

The various peptides can be present with a counterion. Usefulcounterions include salts of acetate, benzenesulfonate, benzoate,calcium edetate, camsylate, carbonate, citrate, edetate (EDTA),edisylate, embonate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, iodide, bromide,chloride, hydroxynaphthoate, isethionate, lactate, lactobionate,estolate, maleate, malate, mandelate, mesylate, mucate, napsylate,nitrate, pantothenate, phosphate, salicylate, stearate, succinate,sulfate, tartarate, theoclate, acetamidobenzoate, adipate, alginate,aminosalicylate, anhydromethylenecitrate, ascorbate, aspartate,camphorate, caprate, caproate, caprylate, cinnamate, cyclamate,dichloroacetate, formate, gentisate, glucuronate, glycerophosphate,glycolate, hippurate, fluoride, malonate, napadisylate, nicotinate,oleate, orotate, oxalate, oxoglutarate, palmitate, pectinate, pectinatepolymer, phenylethylbarbiturate, picrate, propionate, pidolate,sebacate, rhodanide, tosylate, tannate

In a second aspect, the invention also features a therapeutic orprophylactic method comprising administering a composition comprising apurified peptide comprising, consisting essentially or consisting of theamino acid sequence of SEQ ID NO:1. For the treatment ofgastrointestinal disorders, the peptide can be administered orally, byrectal suppository or parenterally.

In various embodiments, the patient is suffering from a gastrointestinaldisorder; the patient is suffering from a disorder selected from thegroup consisting of: a gastrointestinal motility disorder, irritablebowel syndrome, a functional gastrointestinal disorder, gastroesophagealreflux disease, duodenogastric reflux, functional heartburn, dyspepsia,functional dyspepsia, nonulcer dyspepsia, gastroparesis, chronicintestinal pseudo-obstruction, colonic pseudo-obstruction, obesity,congestive heart failure, or benign prostatic hyperplasia; thecomposition is administered orally; the peptide comprises 150, 140, 130,120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 or fewer amino acids. Inother embodiments, the peptide comprises 20 or fewer amino acids, andthe peptide comprises no more than 5 amino acids prior to Cys₄. In otherembodiments the peptide comprises no more than 20, 15, 10, or 5 peptidessubsequent to Cys₁₅. In certain embodiments Xaa₁₆ is a chymotrypsin ortrypsin cleavage site and an analgesic peptide is present immediatelyfollowing Xaa₁₆.

Among the useful peptides are those comprising, consisting of orconsisting essentially of any of the following amino acid sequences:

(SEQ ID NO: 40) SHTCEICAFAACAGC (opossum guanylin); (SEQ ID NO: 4)PGTCEICAYAACTGC (human guanylin); (SEQ ID NO: 41)PSTCEICAYAACAGC (pig guanylin); (SEQ ID NO: 42PNTCEICAYAACTGC (rat guanylin); (SEQ ID NO: 43)PDPCEICANAACTGCL (European eel guanylin, inferred); (SEQ ID NO: 44)NDDCELCVNVACTGCL (human uroguanylin); (SEQ ID NO: 45)QEECELCINMACTGY (opossum lymphoguanylin); (SEQ ID NO: 46)GDDCELCVNVACTGCS (pig uroguanylin); (SEQ ID NO: 47)NDECELCVNIACTGC (guinea pig uroguanylin); (SEQ ID NO: 48)TDECELCINVACTGC (rat uroguanylin); (SEQ ID NO: 49)QEDCELCINVACTGC (opossum uroguanylin); (SEQ ID NO: 50)MPSTQYIRRPASSYASCIWCTTACASCHGRTTKPSLAT (EAST 1); (SEQ ID NO: 51)MPSTQYIRRPASSYASCIWCATACASCHGRTTKPSLAT; (SEQ ID NO: 52)MPSTQYIRRPTSSYASCIWCATACASCHGRTTKPSLAT; (SEQ ID NO: 53)MPSTQYIRRPTSSYASCIWCATVCASCHGRTTKPSLAT; (SEQ ID NO: 54)MPSTQYIRRPASSYASCIWYATACASCHGRTTEPSLAT; (SEQ ID NO: 55)QEECELSINMACTGY (opossum lymphoguanylin analog); (SEQ ID NO: 56)YDECEICMFAACTGC (Japanese eel guanylin); (SEQ ID NO: 57)VCEICAFAACTGC (Zebrafish guanylin, inferred); (SEQ ID NO: 58)ADLCEICAFAACTGCL (Japenese eel renoguanylin, inferred); (SEQ ID NO: 59)PGTCEICAYAACTGCL; (SEQ ID NO: 60) PGTCEICAYAACTGCLKK; (SEQ ID NO: 61)PNTCEICAYAACTGCKKKKKK; (SEQ ID NO: 62) PNTCEICAYAACTGCD; (SEQ ID NO: 63)PNTCEICAYAACTGCDK; (SEQ ID NO: 64) YPNTCEICAYAACTGC; (SEQ ID NO: 65KNTCEICAYAACTGC (SEQ ID NO: 66) KPNTCEICAYAACTGC; (SEQ ID NO: 67)EDPGTCEICAYAACTGC; (SEQ ID NO: 68)VTVQDG NFSFSLESVK KLKDLQEPQE PRVGKLRNFA PIPGEPVVPI LCSNPNFPEELKPLCKEPNA QEILQRLEEIAEDPGTCEICAYAACTGC; (SEQ ID NO: 69)DPGTCEICAYAACTGC; (SEQ ID NO: 70)MNAFLLSALC LLGAWAALAG GVTVQDGNFS FSLESVKKLK DLQEPQEPRVGKLRNFAPIP GEPVVPILCS NPNFPEELKP LCKEPNAQEI LQRLEEIAED PGTCEICAYAACTGC;(SEQ ID NO: 71) MNAFLLFALC LLGAWAALAG GVTVQDGNFS FSLEPRVGKL RNFAPIPGEPVVPILCSNPN FPEELKPLCK EPNAQEILQR LEEIAEDPGTCEICAYAACTGC; (SEQ ID NO: 72)TGSMNAFLLF ALCLLGAWAA LAGGVTVQDG NFSFSLEPRV GKLRNFAPIPGEPVVPILCS NPNFPEELKP LCKEPNAQEI LQRLEEIAEDPGTCEICAYAACTGCLEG;(SEQ ID NO: 73) NDECELCVNVACTGCL; (SEQ ID NO: 74) ECELCVNVACTGCL;(SEQ ID NO: 75) EDCELCINVACTGC; (SEQ ID NO: 76) NDDCELCVACTGCL;(SEQ ID NO: 77) FKTLRTIANDDCELCVNVACTGCL; (SEQ ID NO: 78)FKTLRTIANDDCLCVNVACTGCL; (SEQ ID NO: 79) DDCELCVNVACTGCL;(SEQ ID NO: 80) DCELCVNVACTGCL; (SEQ ID NO: 81) CELCVNVACTGCL;(SEQ ID NO: 82) KDDCELCVNVACTGCL; (SEQ ID NO. 83) PNTCEICANPACTGC.

The peptides can include the amino acid sequence of a peptide thatoccurs naturally in a vertebrate (e.g., mammalian) species or in abacterial species. In addition, the peptides can be partially orcompletely non-naturally occurring peptides.

In a third aspect, the invention features a method for treating apatient suffering from constipation, the method comprising administeringa composition comprising a peptide comprising, consisting essentially orconsisting of the amino acid sequence of SEQ ID NO:1. Clinicallyaccepted criteria that define constipation range from the frequency ofbowel movements, the consistency of feces and the ease of bowelmovement. One common definition of constipation is less than three bowelmovements per week. Other definitions include abnormally hard stools ordefecation that requires excessive straining (Schiller 2001 AlimentPharmacol Ther 15:749-763). Constipation may be idiopathic (functionalconstipation or slow transit constipation) or secondary to other causesincluding neurologic, metabolic or endocrine disorders. These disordersinclude diabetes mellitus, hypothyroidism, hyperthyroidism,hypocalcaemia, Multiple sclerosis, Parkinson's disease, spinal cordlesions, Neurofibromatosis, autonomic neuropathy, Chagas disease,Hirschsprung disease and cystic fibrosis. Constipation may also be theresult of surgery or due to the use of drugs such as analgesics (likeopioids), antihypertensives, anticonvulsants, antidepressants,antispasmodics and antipsychotics.

In various embodiments, the constipation is associated with use of atherapeutic agent; the constipation is associated with a neuropathicdisorder; the constipation is post-surgical constipation; theconstipation is associated with a gastrointestinal disorder; theconstipation is idiopathic (functional constipation or slow transitconstipation); the constipation is associated with neuropathic,metabolic or endocrine disorder (e.g., diabetes mellitus,hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis,Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomicneuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis).Constipation may also be the result of surgery or due to the use ofdrugs such as analgesics (e.g., opioids), antihypertensives,anticonvulsants, antidepressants, antispasmodics and antipsychotics.

In a fourth aspect, the invention features a method for treating apatient suffering a gastrointestinal disorder, the method comprisingadministering to the patient a composition comprising a purified peptidecomprising, consisting essentially of or consisting of the amino acidsequence of SEQ ID NO:1.

In various embodiments, the patient is suffering from a gastrointestinaldisorder; the patient is suffering from a disorder selected from thegroup consisting of: a gastrointestinal motility disorder, irritablebowel syndrome, a functional gastrointestinal disorder, gastroesophagealreflux disease, functional heartburn, dyspepsia, functional dyspepsia,nonulcer dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction, colonic pseudo-obstruction; Crohn's disease,ulcerative colitis, Inflammatory bowel disease, colonicpseudo-obstruction, obesity, congestive heart failure, and benignprostatic hyperplasia.

In a fifth aspect, the invention features a method for increasinggastrointestinal motility in a patient, the method comprisingadministering to the patient a composition comprising a purified peptidecomprising, consisting essentially of or consisting of the amino acidsequence of SEQ ID NO:1.

In a sixth aspect, the invention features a method for decreasinggastrointestinal pain or visceral pain in a patient, the methodcomprising administering to the patient a composition comprising apurified peptide comprising, consisting essentially of or consisting ofthe amino acid sequence of SEQ ID NO:1.

In a seventh aspect, the invention features a method for increasing theactivity of an intestinal guanylate cyclase (GC-C) receptor in apatient, the method comprising administering to the patient acomposition comprising a purified peptide comprising, consistingessentially of or consisting of the amino acid sequence of SEQ ID NO:1.

In an eighth aspect, the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a peptide comprising,consisting essentially of or consisting of the amino acid sequence ofSEQ ID NO:1.

In a ninth aspect, the invention features a composition comprising apurified polypeptide comprising, consisting essentially of or consistingof the amino acid sequence of SEQ ID NO:1. In an embodiment, thecomposition is a pharmaceutical composition.

In a tenth aspect, the invention features a method for treating obesity,the method comprising administering a composition comprising a purifiedpeptide comprising, consisting essentially of or consisting of the aminoacid sequence of SEQ ID NO:1. The peptide can be administered incombination with one or more agents for treatment of obesity, forexample, gut hormone fragment peptide YY₃₋₃₆ (PYY₃₋₃₆) (N Engl. J. Med.349:941, 2003; ikpeapge daspeelnry yaslrhylnl vtrqry) or a variantthereof, glp-1 (glucagon-like peptide-1), exendin-4 (an inhibitor ofglp-1), sibutramine, phentermine, phendimetrazine, benzphetaminehydrochloride (Didrex), orlistat (Xenical), diethylpropion hydrochloride(Tenuate), fluoxetine (Prozac), bupropion, ephedra, chromium, garciniacambogia, benzocaine, bladderwrack (focus vesiculosus), chitosan, nomameherba, galega (Goat's Rue, French Lilac), conjugated linoleic acid,L-carnitine, fiber (psyllium, plantago, guar fiber), caffeine,dehydroepiandrosterone, germander (teucrium chamaedrys),B-hydroxy-O-methylbutyrate, ATL-962 (Alizyme PLC), and pyruvate. Apeptide useful for treating obesity can be administered as a co-therapywith a peptide of the invention either as a distinct molecule or as partof a fusion protein with a peptide of the invention. Thus, for example,PYY₃₋₃₆ can be fused to the carboxy or amino terminus of a peptide ofthe invention. Such a fusion protein can include a chymostrypsin ortrypsin cleavage site that can permit cleavage to separate the twopeptides. A peptide useful for treating obesity can be administered as aco-therapy with electrostimulation (U.S. 20040015201).

In an eleventh aspect, the invention features a method for treatingcongestive heart failure, the method comprising: administering to thepatient a composition comprising a purified peptide comprising,consisting essentially of or consisting of the amino acid sequence ofSEQ ID NO:1. The peptide can be administered in combination with one ormore agents for treatment of congestive heart failure, for example, anatriuretic peptide such as atrial natriuretic peptide, brainnatriuretic peptide or C-type natriuretic peptide), a diuretic, or aninhibitor of angiotensin converting enzyme.

In a twelfth aspect, the invention features a method for treating benignprostatic hyperplasia, the method comprising: administering to thepatient a composition comprising a purified peptide comprising,consisting essentially of or consisting of the amino acid sequence ofSEQ ID NO:1. The peptide can be administered in combination with one ormore agents for treatment of BPH, for example, a 5-alpha reductaseinhibitor (e.g., finasteride) or an alpha adrenergic inhibitor (e.g.,doxazosine).

In a thirteenth aspect, the invention features a method for treating apatient suffering a gastrointestinal disorder, the method comprisingadministering to the patient a composition comprising a complete orpartial agonist of the GC-C receptor. In various embodiments, thepatient is suffering from a gastrointestinal disorder; the patient issuffering from a disorder selected from the group consisting of: agastrointestinal motility disorder, irritable bowel syndrome, afunctional gastrointestinal disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, functional dyspepsia, nonulcerdyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, andcolonic pseudo-obstruction.

In a fourteenth aspect, the invention features a method for treating apatient suffering from constipation, the method comprising administeringa composition comprising a complete or partial agonist of the GC-Creceptor.

In a fifteenth aspect, the invention features a method for increasinggastrointestinal motility in a patient, the method comprisingadministering to the patient a composition comprising a complete orpartial agonist of the GC-C receptor.

In a sixteenth aspect, the invention features a method for decreasinggastrointestinal pain or visceral pain in a patient, the methodcomprising administering to the patient a composition comprising acomplete or partial agonist of the GC-C receptor.

In a seventeenth aspect, the invention features a method for treatingcongestive heart failure, the method comprising administering a completeor partial agonist of the GC-C receptor. GC-C agonists can act in thekidney and adrenal gland to control natriuresis, kaliuresis, anddiuresis thereby reducing the build-up of fluid associated withcongestive heart failure (Lorenz et al. J Clin Invest 112:1138, 2003;Carrithers et al. Kidney Int 65:40, 2004). The agonist can beadministered in combination with one or more agents for treatment ofcongestive heart failure, for example, a natriuretic peptide such asatrial natriuretic peptide, brain natriuretic peptide or C-typenatriuretic peptide), a diuretic, or an inhibitor of angiotensinconverting enzyme.

In an eighteenth aspect, the invention features a method for treatingBPH, the method comprising administering a complete or partial agonistof the GC-C receptor. GC-C agonists acting in the prostate can reducecellular hypertrophy and complications associated with cellularhypertrophy. The agonist can be administered in combination with one ormore agents for treatment of BPH, for example, a 5-alpha reductaseinhibitor (e.g., finasteride) or an alpha adrenergic inhibitor (e.g.,doxazosine).

In a nineteenth aspect, the invention features a method for treatingobesity, the method comprising administering a complete or partialagonist of the GC-C receptor. The agonist can be administered incombination with one or more agents for treatment of obesity, forexample, sibutramine.

The peptides and agonists of the GC-C receptor can be used to treatconstipation or decreased intestinal motility, slow digestion or slowstomach emptying. The peptides can be used to relieve one or moresymptoms of IBS (bloating, pain, constipation), GERD (acid reflux intothe esophagus), duodenogastric reflux, functional dyspepsia, orgastroparesis (nausea, vomiting, bloating, delayed gastric emptying) andother disorders described herein.

Clinically accepted criteria that define constipation range from thefrequency of bowel movements, the consistency of feces and the ease ofbowel movement. One common definition of constipation is less than threebowel movements per week. Other definitions include abnormally hardstools or defecation that requires excessive straining (Schiller 2001,Aliment Pharmacol Ther 15:749-763). Constipation may be idiopathic(functional constipation or slow transit constipation) or secondary toother causes including neurologic, metabolic or endocrine disorders.These disorders include diabetes mellitus, hypothyroidism,hyperthyroidism, hypocalcaemia, Multiple Sclerosis, Parkinson's disease,spinal cord lesions, Neurofibromatosis, autonomic neuropathy, Chagasdisease, Hirschsprung's disease and cystic fibrosis. Constipation mayalso be the result of surgery or due to the use of drugs such asanalgesics (like opioids), antihypertensives, anticonvulsants,antidepressants, antispasmodics and antipsychotics.

In a twentieth aspect, the invention features isolated nucleic acidmolecules comprising or consisting of a sequence encoding a peptide ofthe invention. The invention also features vectors, e.g., expressionvectors that include such nucleic acid molecules and can be used toexpress a peptide of the invention in a cultured cell (e.g., aeukaryotic cell or a prokaryotic cell). The vector can further includeone or more regulatory elements, e.g., a heterologous promoter orelements required for translation operably linked to the sequenceencoding the peptide. In some cases the nucleic acid molecule willencode an amino acid sequence that includes the amino acid sequence of apeptide of the invention. For example, the nucleic acid molecule canencode a preprotein or a preproprotein that can be processed to producea peptide of the invention.

A vector that includes a nucleotide sequence encoding a peptide of theinvention or a peptide or polypeptide comprising a peptide of theinvention may be either RNA or DNA, single- or double-stranded,prokaryotic, eukaryotic, or viral. Vectors can include transposons,viral vectors, episomes, (e.g., plasmids), chromosomes inserts, andartificial chromosomes (e.g. BACs or YACs). Suitable bacterial hosts forexpression of the encode peptide or polypeptide include, but are notlimited to, E. coli. Suitable eukaryotic hosts include yeast such as S.cerevisiae, other fungi, vertebrate cells, invertebrate cells (e.g.,insect cells), plant cells, human cells, human tissue cells, and wholeeukaryotic organisms. (e.g., a transgenic plant or a transgenic animal).Further, the vector nucleic acid can be used to generate a virus such asvaccinia or baculovirus.

As noted above the invention includes vectors and genetic constructssuitable for production of a peptide of the invention or a peptide orpolypeptide comprising such a peptide. Generally, the genetic constructalso includes, in addition to the encoding nucleic acid molecule,elements that allow expression, such as a promoter and regulatorysequences. The expression vectors may contain transcriptional controlsequences that control transcriptional initiation, such as promoter,enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art andmay be functional in, but are not limited to, a bacterium, yeast, plant,or animal cell. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, a poly A addition site, or an internal ribosome entrysite), a splicing sequence or splicing regulatory sequence, and atranscription termination sequence. The vector can be capable ofautonomous replication or it can integrate into host DNA.

The invention also includes isolated host cells harboring one of theforgoing nucleic acid molecules and methods for producing a peptide byculturing such a cell and recovering the peptide or a precursor of thepeptide. Recovery of the peptide or precursor may refer to collectingthe growth solution and need not involve additional steps ofpurification. Proteins of the present invention, however, can bepurified using standard purification techniques, such as, but notlimited to, affinity chromatography, thermaprecipitation, immunoaffinitychromatography, ammonium sulfate precipitation, ion exchangechromatography, filtration, electrophoresis and hydrophobic interactionchromatography.

In a twenty first aspect, the invention features a method of increasingthe level of cyclic guanosine 3′-monophosphate (cGMP) in an organ,tissue (e.g, the intestinal mucosa), or cell (e.g., a cell bearing GC-Areceptor) by administering a composition that includes a peptide of theinvention.

The details of one or more embodiments of the invention are set forth inthe accompanying description and claims. The publications and patentsreferenced herein are incorporated by reference.

DRAWINGS

FIG. 1 depicts deletion variants of human guanylin in which one, two,three or four amino acids are deleted. The deleted amino acids arebetween Cys_(a) and Cys_(d) as well as amino terminal to Cys_(a).

FIG. 2 depicts insertion variants of human guanylin in which one, two,three or four amino acids are inserted. The inserted amino acids arebetween Cys_(a) and Cys_(d) as well as amino terminal to Cys_(a) andcarboxy terminal to Cys_(d).

FIG. 3 depicts various polypeptides which include the amino acidsequence: Xaa₁ Xaa₂ Xaa₃ Cys₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Cys₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ (SEQ ID NO:1) wherein: Xaa₁ is any amino acid oris missing; Xaa₂ is any amino acid or is missing; Xaa₃ is any amino acidor is missing; Xaa₅ is Glu; Xaa₆ is Tyr, Trp, Phe or Leu; Xaa₇ is Cys;Xaa₈ is any of the 20 naturally-occurring amino acids other than Cys oris missing; Xaa₉ is any of the 20 naturally-occurring amino acids; Xaa₁₀is Pro or Gly; Xaa₁₁ is any of the 20 naturally-occurring amino acids;Xaa₁₃ is Thr, Val or Gly; Xaa₁₄ is Gly or Ala; Xaa₁₅ is Cys; and Xaa₁₆is any of the 20 naturally-occurring amino acids or is missing.

DETAILED DESCRIPTION

The peptides of the invention bind to the guanylate cyclase (GC-C)receptor, a key regulator of fluid and electrolyte balance in theintestine and kidney. When stimulated, this receptor, which is locatedon the apical membrane of the intestinal epithelial surface, causes anincrease in intestinal epithelial cyclic GMP (cGMP). This increase incGMP is believed to cause a decrease in water and sodium absorption andan increase in chloride and potassium ion secretion, leading to changesin intestinal fluid and electrolyte transport and increased intestinalmotility. The intestinal GC-C receptor possesses an extracellular ligandbinding region, a transmembrane region, an intracellular proteinkinase-like region and a cyclase catalytic domain. Proposed functionsfor the GC-C receptor are the fluid and electrolyte homeostasis, theregulation of epithelial cell proliferation and the induction ofapoptosis (Shaibhubhai 2002 Curr Opin Drug Dis Devel 5:261-268).

In addition to being expressed in gastrointestinal epithelial cells,GC-C is expressed in extra-intestinal tissues including kidney, lung,pancreas, pituitary, adrenal, developing liver, heart and male andfemale reproductive tissues (reviewed in Vaandrager 2002 Mol CellBiochem 230:73-83). This suggests that the GC-C receptor agonists can beused in the treatment of disorders outside the GI tract, for example,congestive heart failure and benign prostatic hyperplasia.

Ghrelin, a peptide hormone secreted by the stomach, is a key regulatorof appetite in humans. Ghrelin expression levels are regulated byfasting and by gastric emptying. (Kim et al., 2003, Neuroreprt14:1317-20; Gualillo et al., 2003, FEBS Letts 552: 105-9). Thus, byincreasing gastrointestinal motility, GC-C receptor agonists may also beused to regulate obesity.

In humans, the GC-C receptor is activated by guanylin (Gn) (U.S. Pat.No. 5,96,097), uroguanylin (Ugn) (U.S. Pat. No. 5,140,102) andlymphoguanylin (Forte et al. 1999 Endocrinology 140:1800-1806).

Many gastrointestinal disorders, including IBS, are associated withabdominal or visceral pain. Certain of the peptides of the inventioninclude the analgesic or anti-nociceptive tags such as thecarboxy-terminal sequence AspPhe immediately following a Trp, Tyr or Phe(i.e., a chymotrypsin cleavage site) or following Lys or Arg (a trypsincleavage site). Chymotrypsin in the intestinal tract will cleave suchpeptides immediately carboxy terminal to the Trp, Phe or Tyr residue,releasing the dipeptide, AspPhe. This dipeptide has been shown to haveanalgesic activity is animal models (Abdikkahi et al. 2001 Fundam ClinPharmacol 15:117-23; Nikfar et al 1997, 29:583-6; Edmundson et al 1998Clin Pharmacol Ther 63:580-93). In this manner such peptides can treatboth pain and inflammation. Other analgesic peptides can be present atthe carboxy terminus of the peptide (following a cleavage site)including: endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron,and substance P. As described in greater detail below, various analgesicpeptides and compounds can be covalently linked to or used incombination therapy with the therapeutic peptides described herein.

In the human body an inactive form of chymotrypsin, chymotrypsinogen isproduced in the pancreas. When this inactive enzyme reaches the smallintestine it is converted to active chymotrypsin by the excision of twodi-peptides. Active chymotrypsin will cleave peptides at the peptidebond on the carboxy-terminal side of Trp, Tyr or Phe. The presence ofactive chymotrypsin in the intestinal tract will lead to cleavage ofcertain of the peptides of the invention having an appropriatelypositioned chymotrypsin cleavage site. Certain of the peptides of theinvention include a Trp, Tyr or Phe immediately followed by acarboxy-terminal analgesic peptide. It is expected that chymotrypsincleavage will release the analgesic peptide from peptide of theinvention having an appropriately positioned chymotrypsin cleavage siteas the peptide passes through the intestinal tract.

Trypsinogen, like chymotrypsin, is a serine protease that is produced inthe pancreas and is present in the digestive tract. The active form,trypsin, will cleave peptides having a Lys or Arg. The presence ofactive trypsin in the intestinal tract will lead to cleavage of certainof the peptides of the invention having an appropriately positionedtrypsin cleavage site. It is expected that chymotrypsin cleavage willrelease the analgesic peptide from peptide of the invention having anappropriately positioned trypsin cleavage site as the peptide passesthrough the intestinal tract.

In some cases, the peptides of the invention are produced as a preproprotein. The prepro protein can include any suitable prepro sequence,including, for example, mnafllsalc llgawaalag gvtvqdgnfs fslesvkklkdlqepqepry gklrnfapip gepvvpilcs npnfpeelkp lckepnaqei lqrleeiaed (SEQID NO:) and mgcraasgll pgvavvllll lqstqsvyiq yqgfrvqles mkklsdleaqwapsprlqaq sllpavchhp alpqdlqpvc asqeassifk tlrtia (SEQ ID NO:) or abacterial leader sequence such as:mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmn. Where thepeptide is produced by a bacterial cell, e.g., E. coli, the forgoingleader sequence will be cleaved and the mature peptide will beefficiently secreted from the bacterial cell. U.S. Pat. No. 5,395,490describes vectors, expression systems and methods for the efficientproduction of certain mature peptides having disulfide bonds inbacterial cells and methods for achieving efficient secretion of suchmature peptides. The vectors, expression systems and methods describedin U.S. Pat. No. 5,395,490 can be used to produce the polypeptides ofthe present invention.

Variant Peptides

The invention includes variant peptides that can include one, two,three, four, or five or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, or15) amino acid substitutions compared to any of the peptides describedabove. The substitution(s) can be conservative or non-conservative. Thenaturally-occurring amino acids can be substituted by D-isomers of anyamino acid, non-natural amino acids, natural and non-natural amino acidanalogs, and other groups. A conservative amino acid substitutionresults in the alteration of an amino acid for a similar acting aminoacid, or amino acid of like charge, polarity, or hydrophobicity. At somepositions, even conservative amino acid substitutions can reduce theactivity of the peptide. A conservative substitution can substitute anaturally-occurring amino acid for a non-naturally-occurring amino acid.Among the naturally occurring amino acid substitutions generallyconsidered conservative are:

For Amino Acid Code Replace with any of Alanine Ala Gly, Cys, SerArginine Arg Lys, His Asparagine Asn Asp, Glu, Gln, Aspartic Acid AspAsn, Glu, Gln Cysteine Cys Met, Thr, Ser Glutamine Gln Asn, Glu, AspGlutamic Acid Glu Asp, Asn, Gln Glycine Gly Ala Histidine His Lys, ArgIsoleucine Ile Val, Leu, Met Leucine Leu Val, Ile, Met Lysine Lys Arg,His Methionine Met Ile, Leu, Val Phenylalanine Phe Tyr, His, Trp ProlinePro Serine Ser Thr, Cys, Ala Threonine Thr Ser, Met, Val Tryptophan TrpPhe, Tyr Tyrosine Tyr Phe, His Valine Val Leu, Ile, Met

In some circumstances it can be desirable to treat patients with avariant peptide that binds to and activates intestinal GC-C receptor,but is less active or more active than the non-variant form of thepeptide. Reduced activity can arise from reduced affinity for thereceptor or a reduced ability to activate the receptor once bound orreduced stability of the peptide. Increased activity can arise fromincreased affinity for the receptor or an increased ability to activatethe receptor once bound or increased stability of the peptide.

In some peptides one or both members of one or both pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, 3-mercaptoproline (Kolodziej et al. 1996 Int J PeptProtein Res 48:274); 4, a dimethylcysteine (Hunt et al. 1993 Int J PeptProtein Res 42:249) or diaminopropionic acid (Smith et al. 1978 J MedChem 21:117) to form alternative internal cross-links at the positionsof the normal disulfide bonds.

Production of Peptides

Useful peptides can be produced either in bacteria including, withoutlimitation, E. coli, or in other existing systems for peptide or proteinproduction (e.g., Bacillus subtilis, baculovirus expression systemsusing Drosophila Sf9 cells, yeast or filamentous fungal expressionsystems, mammalian cell expression systems), or they can be chemicallysynthesized.

If the peptide or variant peptide is to be produced in bacteria, e.g.,E. coli, the nucleic acid molecule encoding the peptide may also encodea leader sequence that permits the secretion of the mature peptide fromthe cell. Thus, the sequence encoding the peptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST peptide. The secreted, mature peptide can be purified fromthe culture medium.

The sequence encoding a peptide of the invention is can be inserted intoa vector capable of delivering and maintaining the nucleic acid moleculein a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B subtilis, Pseudomonas, Salmonella. The geneticconstruct also includes, in addition to the encoding nucleic acidmolecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art.The expression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or aninternal ribosome entry site). The vector can be capable of autonomousreplication or it can integrate into host DNA to ensure stability duringpeptide production.

The protein coding sequence that includes a peptide of the invention canalso be fused to a nucleic acid encoding a polypeptide affinity tag,e.g., glutathione S-transferase (GST), maltose E binding protein,protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag, inorder to facilitate purification. The affinity tag or reporter fusionjoins the reading frame of the peptide of interest to the reading frameof the gene encoding the affinity tag such that a translational fusionis generated. Expression of the fusion gene results in translation of asingle polypeptide that includes both the peptide of interest and theaffinity tag. In some instances where affinity tags are utilized, DNAsequence encoding a protease recognition site will be fused between thereading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the peptides and variants of the invention in proteinexpression systems other than bacteria, and well known to those skilledin the art, can also be used to produce peptides in a biological system.

Mature peptides and variants thereof can be synthesized by thesolid-phase method using an automated peptide synthesizer. For example,the peptide can be synthesized on Cyc(4-CH₂Bxl)-OCH₂-4-(oxymethyl)-phenylacetamidomethyl resin using a doublecoupling program. Protecting groups must be used appropriately to createthe correct disulfide bond pattern. For example, the followingprotecting groups can be used: t-butyloxycarbonyl (alpha-amino groups);acetamidomethyl (thiol groups of Cys residues B and E); 4-methylbenyl(thiol groups of Cys residues C and F); benzyl (y-carboxyl of glutamicacid and the hydroxyl group of threonine, if present); and bromobenzyl(phenolic group of tyrosine, if present). Coupling is effected withsymmetrical anhydride of t-butoxylcarbonylamino acids orhydroxybenzotriazole ester (for asparagine or glutamine residues), andthe peptide is deprotected and cleaved from the solid support inhydrogen fluoride, dimethyl sulfide, anisole, and p-thiocresol using8/1/1/0.5 ratio (v/v/v/w) at 0° C. for 60 min. After removal of hydrogenfluoride and dimethyl sulfide by reduced pressure and anisole andp-thiocresol by extraction with ethyl ether and ethyl acetatesequentially, crude peptides are extracted with a mixture of 0.5M sodiumphosphate buffer, pH 8.0 and N,N-dimethylformamide using 1/1 ratio, v/v.The disulfide bond for Cys residues B and E is the formed using dimethylsulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62). Theresulting peptide is the purified by reverse-phase chromatography. Thedisulfide bond between Cys residues C and F is formed by firstdissolving the peptide in 50% acetic acid in water. Saturated iodinesolution in glacial acetic acid is added (1 ml iodine solution per 100ml solution). After incubation at room temperature for 2 days in anenclosed glass container, the solution is diluted five-fold withdeionized water and extracted with ethyl ether four times for removal ofunreacted iodine. After removal of the residual amount of ethyl ether byrotary evaporation the solution of crude product is lyophilized andpurified by successive reverse-phase chromatography.

Intestinal GC-C Receptor Binding and Activity Assays

The ability of peptides, variant peptides and other compounds to bind toand activate the intestinal GC-C receptor can be tested using the T84human colon carcinoma cell line (American Type Culture Collection(Bethesda, Md.).

Briefly, cells are grown to confluency in 24-well culture plates with a1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium(DMEM), supplemented with 5% fetal calf serum and are used at betweenpassages 54 and 60.

Monolayers of T84 cells in 24-well plates are washed twice with 1ml/well DMEM, then incubated at 37° C. for 10 min with 0.45 ml DMEMcontaining 1 mM isobutylmethylxanthine (IBMX), a cyclic nucleotidephosphodiesterase inhibitor. Test peptides (50T1) are then added andincubated for 30 minutes at 37° C. The media is aspirated and thereaction is terminated by the addition of ice cold 0.5 ml of 0.1N HCl.The samples are held on ice for 20 minutes and then evaporated todryness using a heat gun or vacuum centrifugation. The dried samples areresuspended in 0.5 ml of phosphate buffer provided in the CaymanChemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, Mich.). CyclicGMP is measured by EIA according to procedures outlined in the CaymanChemical Cyclic GMP EIA kit.

For the binding assay, T84 cell monolayers in 24-well plates are washedtwice with 1 ml of binding buffer (DMEM containing 0.05% bovine serumalbumin and 25 mM HEPES, pH 7.2), then incubated for 30 min at 37° C. inthe presence of mature radioactively labeled E. coli ST peptide and thetest material at various concentrations. The cells are then washed 4times with 1 ml of DMEM and solubilized with 0.5 ml/well 1N NaOH. Thelevel of radioactivity in the solubilized material is then determinedusing standard methods.

Murine Gastrointestinal Transit (GIT) Assay

In order to determine whether a test compound or a peptide, increasesthe rate of gastrointestinal transit, the test compound can be tested inthe murine gastrointestinal transit (GIT) assay (Moon et al. Infectionand Immunity 25:127, 1979). In this assay, charcoal, which can bereadily visualized in the gastrointestinal tract is administered to miceafter the administration of a test compound. The distance traveled bythe charcoal is measured and expressed as a percentage of the totallength of the colon.

Mice are fasted with free access to water for 12 to 16 hours before thetreatment with peptide or control buffer. The peptides are orallyadministered at 1 Tg/kg-1 mg/kg of peptide in buffer (20 mM Tris pH 7.5)seven minutes before being given an oral dose of 5% Activated Carbon(Aldrich 242276-250G). Control mice are administered buffer only beforebeing given a dose of Activated Carbon. After 15 minutes, the mice aresacrificed and their intestines from the stomach to the cecum aredissected. The total length of the intestine as well as the distancetraveled from the stomach to the charcoal front is measured for eachanimal and the results are expressed as the percent of the total lengthof the intestine traveled by the charcoal front. Results are reported asthe average of 10 mice±standard deviation. A comparison of the distancetraveled by the charcoal between the mice treated with peptide versusthe mice treated with vehicle alone is performed using a Student's ttest and a statistically significant difference is considered forP<0.05. Positive controls for this assay may include commerciallyavailable wild-type ST peptide (Sigma-Aldrich, St Louis, Mo.) andZelnorm®, a drug approved for IBS that is an agonist for the serotoninreceptor 5HT4.

Suckling Mouse Model of Intestinal Secretion (SuMi Assay)

The peptides of the invention can be tested for their ability toincrease intestinal secretion using a suckling mouse model of intestinalsecretion. In this model a test compound is administered to sucklingmice that are between seven and nine days old. After the mice aresacrificed, the gastrointestinal tract from the stomach to the cecum isdissected (“guts”). The remains (“carcass”) as well as the guts areweighed and the ratio of guts to carcass weight is calculated. If theratio is above 0.09, one can conclude that the test compound increasesintestinal secretion. Controls for this assay may include wild-type STpeptide and Zelnorm®

Phenylbenzoquinone-Induced Writhing Model

The PBQ-induced writhing model can be used to assess pain controlactivity of the peptides and GC-C receptor agonists of the invention.This model is described by Siegmund et al. (1957 Proc. Soc. Exp. Bio.Med. 95:729-731). Briefly, one hour after oral dosing with a testcompound, e.g., a peptide, morphine or vehicle, 0.02% phenylbenzoquinone(PBQ) solution (12.5 mL/kg) is injected by intraperitoneal route intothe mouse. The number of stretches and writhings are recorded from the5^(th) to the 10^(th) minute after PBQ injection, and can also becounted between the 35^(th) and 40^(th) minute and between the 60^(th)and 65^(th) minute to provide a kinetic assessment. The results areexpressed as the number of stretches and writhings (mean±SEM) and thepercentage of variation of the nociceptive threshold calculated from themean value of the vehicle-treated group. The statistical significance ofany differences between the treated groups and the control group isdetermined by a Dunnett's test using the residual variance after aone-way analysis of variance (P<0.05) using SigmaStat Software.

Colonic Hyperalgesia Animal Models

Hypersensitivity to colorectal distension is a common feature inpatients with IBS and may be responsible for the major symptom of pain.Both inflammatory and non-inflammatory animal models of visceralhyperalgesia to distension have been developed to investigate the effectof compounds on visceral pain in IBS.

I. Trinitrobenzenesulphonic Acid (TNBS)-Induced Rectal Allodynia Model

Male Wistar rats (220-250 g) are premedicated with 0.5 mg/kg ofacepromazine injected intraperitoneally (IP) and anesthetized byintramuscular administration of 100 mg/kg of ketamine. Pairs of nichromewire electrodes (60 cm in length and 80 μm in diameter) are implanted inthe striated muscle of the abdomen, 2 cm laterally from the white line.The free ends of electrodes are exteriorized on the back of the neck andprotected by a plastic tube attached to the skin. Electromyographic(EMG) recordings are started 5 days after surgery. Electrical activityof abdominal striated muscle is recorded with an electroencephalographmachine (Mini VIII, Alvar, Paris, France) using a short time constant(0.03 sec.) to remove low-frequency signals (<3 Hz).

Ten days post surgical implantation, trinitrobenzenesulphonic acid(TNBS) is administered to induce rectal inflammation. TNBS (80 mg kg⁻¹in 0.3 ml 50% ethanol) is administered intrarectally through a siliconerubber catheter introduced at 3 cm from the anus under lightdiethyl-ether anesthesia, as described (Morteau et al. 1994 Dig Dis Sci39:1239). Following TNBS administration, rats are placed in plastictunnels where they are severely limited in mobility for several daysbefore colorectal distension (CRD). Experimental compound isadministered one hour before CRD which is performed by insertion intothe rectum, at 1 cm of the anus, a 4 cm long balloon made from a latexcondom (Gue et al, 1997 Neurogastroenterol. Motil. 9:271). The balloonis fixed on a rigid catheter taken from an embolectomy probe (Fogarty).The catheter attached balloon is fixed at the base of the tail. Theballoon, connected to a barostat is inflated progressively by step of 15mmHg, from 0 to 60 mmHg, each step of inflation lasting 5 min.Evaluation of rectal sensitivity, as measured by EMG, is performedbefore (1-2 days) and 3 days following rectal instillation of TNBS.

The number of spike bursts that corresponds to abdominal contractions isdetermined per 5 min periods. Statistical analysis of the number ofabdominal contractions and evaluation of the dose-effects relationshipsis performed by a one way analysis of variance (ANOVA) followed by apost-hoc (Student or Dunnett tests) and regression analysis for ED50 ifappropriate.

II. Stress-Induced Hyperalgesia Model

Male Wistar Rats (200-250 g) are surgically implanted with nichrome wireelectrodes as in the TNBS model. Ten days post surgical implantation,partial restraint stress (PRS), is performed as described by Williams etal. for two hours (Williams et al. 1988 Gastroenterology 64:611).Briefly, under light anaesthesia with ethyl-ether, the foreshoulders,upper forelimbs and thoracic trunk are wrapped in a confining harness ofpaper tape to restrict, but not prevent body movements. Controlsham-stress animals are anaesthetized but not wrapped. Thirty minutesbefore the end of the PRS session, the animals are administeredtest-compound or vehicle. Thirty minutes to one hour after PRScompletion, the CRD distension procedure is performed as described abovefor the TNBS model with barostat at pressures of 15, 30, 45 and 60 mmHg. Statistical analysis on the number of bursts is determined andanalyzed as in the TNBS model above.

Administration of Peptides and GC-C Receptor Agonists

For treatment of gastrointestinal disorders, the peptides and agonistsof the invention are can be administered orally, e.g., as a tablet orcachet containing a predetermined amount of the active ingredient,pellet, gel, paste, syrup, bolus, electuary, slurry, capsule; powder;granules; as a solution or a suspension in an aqueous liquid or anon-aqueous liquid; as an oil-in-water liquid emulsion or a water-in-oilliquid emulsion, via a liposomal formulation (see, e.g., EP 736299) orin some other form. Orally administered compositions can includebinders, lubricants, inert diluents, lubricating, surface active ordispersing agents, flavoring agents, and humectants. Orally administeredformulations such as tablets may optionally be coated or scored and maybe formulated so as to provide sustained, delayed or controlled releaseof the active ingredient therein. The peptides and agonists can beco-administered with other agents used to treat gastrointestinaldisorders including but not limited to acid suppressing agents such asHistamine-2 receptor agonists (H2As) and proton pump inhibitors (PPIs).The peptides and agonists can also be administered by rectalsuppository. For the treatment of disorders outside the gastrointestinaltract such as congestive heart failure and benign prostatic hypertrophy,peptides and agonists can be administered parenterally or orally.

The peptides described herein can be used alone or in combination withother agents. For example, the peptides can be administered togetherwith one or more analgesic peptides or compounds. The analgesic peptideand/or compound can be covalently attached to a peptide described hereinor it can be a separate agent that is administered together with orsequentially with a peptide described herein in a combination therapy.

Combination therapy can be achieved by administering two or more agents,e.g., a peptide described herein and an analgesic peptide or compound,each of which is formulated and administered separately, or byadministering two or more agents in a single formulation. Othercombinations are also encompassed by combination therapy. For example,two agents can be formulated together and administered in conjunctionwith a separate formulation containing a third agent. While the two ormore agents in the combination therapy can be administeredsimultaneously, they need not be. For example, administration of a firstagent (or combination of agents) can precede administration of a secondagent (or combination of agents) by minutes, hours, days, or weeks.Thus, the two or more agents can be administered within minutes of eachother or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other orwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other orwithin 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some caseseven longer intervals are possible. While in many cases it is desirablethat the two or more agents used in a combination therapy be present inwithin the patient's body at the same time, this need not be so.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

The agents, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium. Thus, they can becombined with materials that do not produce an adverse, allergic orotherwise unwanted reaction when administered to a patient. The carriersor mediums used can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose, diluents, lubricants, binders,disintegrating agents, and the like), etc. If desired, tablet dosages ofthe disclosed compositions may be coated by standard aqueous ornonaqueous techniques.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, and thelike. Any such optional ingredient must be compatible with the compoundof the invention to insure the stability of the formulation. Thecomposition may contain other additives as needed, including for exanplelactose, glucose, fructose, galactose, trehalose, sucrose, maltose,raffinose, maltitol, melezitose, stachyose, lactitol, palatinite,starch, xylitol, mannitol, myoinositol, and the like, and hydratesthereof, and amino acids, for example alanine, glycine and betaine, andpeptides and proteins, for example albumen.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents such as:

BINDERS: corn starch, potato starch, other starches, gelatin, naturaland synthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500LM®, sold by Colorcon, Ltd.), hydroxypropyl methyl cellulose,microcrystalline cellulose (e.g. AVICEL™, such as, AVICEL-PH-101 ™,-103™ and -105TH, sold by FMC Corporation, Marcus Hook, Pa., USA), ormixtures thereof,

FILLERS: talc, calcium carbonate (e.g., granules or powder), dibasiccalcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g.,granules or powder), microcrystalline cellulose, powdered cellulose,dextrates, kaolin, mannitol, silicic acid, sorbitol, starch,pre-gelatinized starch, or mixtures thereof,

DISINTEGRANTS: agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, clays, other algins, othercelluloses, gums, or mixtures thereof,

LUBRICANTS: calcium stearate, magnesium stearate, mineral oil, lightmineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, otherglycols, stearic acid, sodium lauryl sulfate, talc, hydrogenatedvegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesameoil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate,ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co.,Baltimore, Md. USA), a coagulated aerosol of synthetic silica (DeaussaCo., Plano, Tex. USA), a pyrogenic silicon dioxide (CAB-O-SIL, CabotCo., Boston, Mass. USA), or mixtures thereof,

ANTI-CAKING AGENTS: calcium silicate, magnesium silicate, silicondioxide, colloidal silicon dioxide, talc, or mixtures thereof,

ANTIMICROBIAL AGENTS: benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride,cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben,phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate,phenylmercuric nitrate, potassium sorbate, propylparaben, sodiumbenzoate, sodium dehydroacetate, sodium propionate, sorbic acid,thimersol, thymo, or mixtures thereof, and

COATING AGENTS: sodium carboxymethyl cellulose, cellulose acetatephthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl cellulosephthalate, methylcellulose, polyethylene glycol, polyvinyl acetatephthalate, shellac, sucrose, titanium dioxide, carnauba wax,microcrystalline wax, or mixtures thereof.

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(M-caprolactone) and poly(alkylene oxide) (U.S.20030068384) to create a sustained release formulation. Suchformulations can be used to implants that release a peptide or anotheragent over a period of a few days, a few weeks or several monthsdepending on the polymer, the particle size of the polymer, and the sizeof the implant (see, e.g., U.S. Pat. No. 6,620,422). Other sustainedrelease formulations and polymers for use in such formulations aredescribed in EP 0 467 389 A2, WO 93/24150, U.S. Pat. No. 5,612,052, WO97/40085, WO 03/075887, WO 01/01964A2, U.S. Pat. No. 5,922,356, WO94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat. No. 5,968,895, U.S.Pat. No. 6,180,608, U.S. 20030171296, U.S. 20020176841, U.S. Pat. No.5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat. No. 5,134,122, U.S. Pat.No. 5,192,741, U.S. Pat. No. 5,192,741, U.S. Pat. No. 4,668,506, U.S.Pat. No. 4,713,244, U.S. Pat. No. 5,445,832 U.S. Pat. No. 4,931,279,U.S. Pat. No. 5,980,945, WO 02/058672, WO 9726015, WO 97/04744, and.US20020019446. In such sustained release formulations microparticles ofpeptide are combined with microparticles of polymer. One or moresustained release implants can be placed in the large intestine, thesmall intestine or both. U.S. Pat. No. 6,011,011 and WO 94/06452describe a sustained release formulation providing either polyethyleneglycols (i.e. PEG 300 and PEG 400) or triacetin. WO 03/053401 describesa formulation which may both enhance bioavailability and providecontrolled release of the agent within the GI tract. Additionalcontrolled release formulations are described in WO 02/38129, EP 326151, U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029; U.S.20030064105, U.S. 20030138488A1, U.S. 20030216307A1,U.S. Pat. No.6,667,060, WO 01/49249, WO 01/49311, WO 01/49249, WO 01/49311, and U.S.Pat. No. 5,877,224.

The agents can be administered, e.g., by intravenous injection,intramuscular injection, subcutaneous injection, intraperitonealinjection, topical, sublingual, intraarticular (in the joints),intradermal, buccal, ophthalmic (including intraocular), intranasaly(including using a cannula), or by other routes. The agents can beadministered orally, e.g., as a tablet or cachet containing apredetermined amount of the active ingredient, gel, pellet, paste,syrup, bolus, electuary, slurry, capsule, powder, granules, as asolution or a suspension in an aqueous liquid or a non-aqueous liquid,as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion,via a micellar formulation (see, e.g. WO 97/11682) via a liposomalformulation (see, e.g., EP 736299,WO 99/59550 and WO 97/13500), viaformulations described in WO 03/094886 or in some other form. Orallyadministered compositions can include binders, lubricants, inertdiluents, lubricating, surface active or dispersing agents, flavoringagents, and humectants. Orally administered formulations such as tabletsmay optionally be coated or scored and may be formulated so as toprovide sustained, delayed or controlled release of the activeingredient therein. The agents can also be administered transdermally(i.e. via reservoir-type or matrix-type patches, microneedles, thermalporation, hypodermic needles, iontophoresis, electroporation, ultrasoundor other forms of sonophoresis, jet injection, or a combination of anyof the preceding methods (Prausnitz et al. 2004, Nature Reviews DrugDiscovery 3:115-124)). The agents can be administered usinghigh-velocity transdermal particle injection techniques using thehydrogel particle formulation described in U.S. 20020061336. Additionalparticle formulations are described in WO 00/45792, WO 00/53160, and WO02/19989. An example of a transdermal formulation containing plaster andthe absorption promoter dimethylisosorbide can be found in WO 89/04179.WO 96/11705 provides formulations suitable for transdermaladministration. The agents can be administered in the form a suppositoryor by other vaginal or rectal means. The agents can be administered in atransmembrane formulation as described in WO 90/07923. The agents can beadministered non-invasively via the dehydrated particles described inU.S. Pat. No. 6,485,706. The agent can be administered in anenteric-coated drug formulation as described in WO 02/49621. The agentscan be administered intranassaly using the formulation described in U.S.Pat. No. 5,179,079. Formulations suitable for parenteral injection aredescribed in WO 00/62759. The agents can be administered using thecasein formulation described in U. S. 20030206939 and WO 00/06108. Theagents can be administered using the particulate formulations describedin U.S. 20020034536.

The agents, alone or in combination with other suitable components, canbe administered by pulmonary route utilizing several techniquesincluding but not limited to intratracheal instillation (delivery ofsolution into the lungs by syringe), intratracheal delivery ofliposomes, insufflation (administration of powder formulation by syringeor any other similar device into the lungs) and aerosol inhalation.Aerosols (e.g., jet or ultrasonic nebulizers, metered-dose inhalers(MDIs), and dry-powder inhalers (DPIs)) can also be used in intranasalapplications. Aerosol formulations are stable dispersions or suspensionsof solid material and liquid droplets in a gaseous medium and can beplaced into pressurized acceptable propellants, such ashydrofluroalkanes (HFAs, i.e. HFA-134a and HFA-227, or a mixturethereof), dichlorodifluoromethane (or other chlorofluocarbon propellantssuch as a mixture of Propellants 11, 12, and/or 114), propane, nitrogen,and the like. Pulmonary formulations may include permeation enhancerssuch as fatty acids, and saccharides, chelating agents, enzymeinhibitors (e.g., protease inhibitors), adjuvants (e.g., glycocholate,surfactin, span 85, and nafamostat), preservatives (e.g., benzalkoniumchloride or chlorobutanol), and ethanol (normally up to 5% but possiblyup to 20%, by weight). Ethanol is commonly included in aerosolcompositions as it can improve the function of the metering valve and insome cases also improve the stability of the dispersion. Pulmonaryformulations may also include surfactants which include but are notlimited to bile salts and those described in U.S. Pat. No. 6,524,557 andreferences therein. The surfactants described in U.S. Pat. No.6,524,557, e.g., a C8-C16 fatty acid salt, a bile salt, a phospholipid,or alkyl saccaride are advantageous in that some of them also reportedlyenhance absorption of the peptide in the formulation. Also suitable inthe invention are dry powder formulations comprising a therapeuticallyeffective amount of active compound blended with an appropriate carrierand adapted for use in connection with a dry-powder inhaler. Absorptionenhancers which can be added to dry powder formulations of the presentinvention include those described in U.S. Pat. No. 6,632,456. WO02/080884 describes new methods for the surface modification of powders.Aerosol formulations may include U.S. Pat. No. 5,230,884, U.S. Pat. No.5,292,499, WO 017/8694, WO 01/78696, U.S. 2003019437, U.S. 20030165436,and WO 96/40089 (which includes vegetable oil). Sustained releaseformulations suitable for inhalation are described in U.S.20010036481A1, 20030232019A1, and U.S. 20040018243A1 as well as in WO01/13891, WO 02/067902, WO 03/072080, and WO 03/079885. Pulmonaryformulations containing microparticles are described in WO 03/015750,U.S. 20030008013, and WO 00/00176. Pulmonary formulations containingstable glassy state powder are described in U.S. 20020141945 and U.S.Pat. No. 6,309,671. Other aerosol formulations are described in EP1338272A1 WO 90/09781, U.S. Pat. No. 5,348,730, U.S. Pat. No. 6,436,367,WO 91/04011, and U.S. Pat. No. 6,294,153 and U.S. Pat. No. 6,290,987describes a liposomal based formulation that can be administered viaaerosol or other means. Powder formulations for inhalation are describedin U.S. 20030053960 and WO 01/60341. The agents can be administeredintranasally as described in U.S. 20010038824.

Solutions of medicament in buffered saline and similar vehicles arecommonly employed to generate an aerosol in a nebulizer. Simplenebulizers operate on Bernoulli's principle and employ a stream of airor oxygen to generate the spray particles. More complex nebulizersemploy ultrasound to create the spray particles. Both types are wellknown in the art and are described in standard textbooks of pharmacysuch as Sprowls' American Pharmacy and Remington's The Science andPractice of Pharmacy. Other devices for generating aerosols employcompressed gases, usually hydrofluorocarbons and chlorofluorocarbons,which are mixed with the medicament and any necessary excipients in apressurized container, these devices are likewise described in standardtextbooks such as Sprowls and Remington.

The agents can be a free acid or base, or a pharmacologically acceptablesalt thereof. Solids can be dissolved or dispersed immediately prior toadministration or earlier. In some circumstances the preparationsinclude a preservative to prevent the growth of microorganisms. Thepharmaceutical forms suitable for injection can include sterile aqueousor organic solutions or dispersions which include, e.g., water, analcohol, an organic solvent, an oil or other solvent or dispersant(e.g., glycerol, propylene glycol, polyethylene glycol, and vegetableoils). The formulations may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Pharmaceutical agents can besterilized by filter sterilization or by other suitable means.

The agent can be fused to immunoglobulins or albumin, or incorporatedinto a lipsome to improve half-life. The agent can also be conjugated topolyethylene glycol (PEG) chains.

Methods for pegylation and additional formulations containingPEG-conjugates (i.e. PEG-based hydrogels, PEG modified liposomes) can befound in Harris and Chess, Nature Reviews Drug Discovery 2: 214-221 andthe references therein. The peptides of the invention may also beconjugated to, for example, alkyl groups (e.g., C1-C20 straight orbranched alkyl groups); fatty acid radicals; and combinations of PEG;alkyl groups and fatty acid radicals (see U.S. Pat. No. 6,309,633;Soltero et al., 2001 Innovations in Pharmaceutical Technology 106-110).The agent can be administered via a nanocochleate or cochleate deliveryvehicle (BioDelivery Sciences International). The agents can bedelivered transmucosally (i.e. across a mucosal surface such as thevagina, eye or nose) using formulations such as that described in U.S.Pat. No. 5,204,108. The agents can be formulated in microcapsules asdescribed in WO 88/01165. The agent can be administered intra-orallyusing the formulations described in U.S. 20020055496, WO 00/47203, andU.S. Pat. No. 6,495,120. The agent can be delivered using nanoemulsionformulations described in WO 01/91728A2.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the active compound(s) with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art, as exemplified by Remington's Pharmaceutical Sciences (18thEdition, Mack Publishing Company, 1995).

The agents described herein and combination therapy agents can bepackaged as a kit that includes single or multiple doses of two or moreagents, each packaged or formulated individually, or single or multipledoses of two or more agents packaged or formulated in combination. Thus,one or more agents can be present in first container, and the kit canoptionally include one or more agents in a second container. Thecontainer or containers are placed within a package, and the package canoptionally include administration or dosage instructions. A kit caninclude additional components such as syringes or other means foradministering the agents as well as diluents or other means forformulation.

Methods to increase chemical and/or physical stability of the agents thedescribed herein are found in U.S. Pat. No. 6,541,606, U.S. Pat. No.6,068,850, U.S. Pat. No. 6,124,261, U.S. Pat. No. 5,904,935, and WO00/15224, U.S. 20030069182 (via the additon of nicotinamide), U.S.20030175230A1, U.S. 20030175230A1, U.S. 20030175239A1, U.S. 20020045582,U.S. 20010031726, WO 02/26248, WO 03/014304, WO 98/00152A1, WO98/00157A1, WO 90/12029, WO 00/04880, and WO 91/04743, WO 97/04796 andthe references cited therein.

Methods to increase bioavailability of the agents described herein arefound in U.S. Pat. No. 6,008,187, U.S. Pat. No. 5,424,289, U.S.20030198619, WO 90/01329, WO 01/49268, WO 00/32172, and WO 02/064166.Glycyrrhizinate can also be used as an absorption enhancer (see, e.g.,EP397447). WO 03/004062 discusses Ulex europaeus I (UEAI) and UEAImimetics which may be used to target the agents of the invention to theGI tract.

Analgesic Agents

The peptides described herein can be used in combination therapy with ananalgesic agent, e.g., an analgesic compound or an analgesic peptide.The analgesic agent can optionally be covalently attached to a peptidedescribed herein. Among the useful analgesic agents are: Ca channelblockers, 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1receptor antagonists), opioid receptor agonists (loperamide, fedotozine,and fentanyl), NK1 receptor antagonists, CCK receptor agonists (e.g.,loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists,norepinephrine-serotonin reuptake inhibitors (NSRI), vanilloid andcannabanoid receptor agonists, and sialorphin. Analgesics agents in thevarious classes are described in the literature.

Among the useful analgesic peptides are sialorphin-related peptides,including those comprising the amino acid sequence QHNPR (SEQ ID NO:85),including: VQHNPR (SEQ ID NO:86); VRQHNPR (SEQ ID NO:87); VRGQHNPR (SEQID NO:88); VRGPQHNPR (SEQ ID NO:89); VRGPRQHNPR (SEQ ID NO:90);VRGPRRQHNPR (SEQ ID NO:91); and RQHNPR (SEQ ID NO:92).Sialorphin-related peptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or peptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the peptides of theinvention in a co-therapy or linked to the peptides of the invention,e.g., by a covalent bond. Sialophin and related peptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

Opioid receptor antagonists and agonists can be administered with thepeptides of the invention in co-therapy or linked to the peptide of theinvention, e.g., by a covalent bond. For example, opioid receptorantagonists such as naloxone, naltrexone, methyl nalozone, nalmefene,cypridime, beta funaltrexamine, naloxonazine, naltrindole, andnor-binaltorphimine are thought to be useful in the treatment of IBS. Itcan be useful to formulate opioid antagonists of this type is a delayedand sustained release formulation such that initial release of theantagonist is in the mid to distal small intestine and/or ascendingcolon. Such antagonists are described in WO 01/32180 A2. Enkephalinpentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist ofthe mu and delta opioid receptors and is thought to be useful forincreasing intestinal motility (Eur. J. Pharm. 219:445, 1992), and thispeptide can be used in conjunction with the peptides of the invention.Also useful is trimebutine which is thought to bind to mu/delta/kappaopioid receptors and activate release of motilin and modulate therelease of gastrin, vasoactive intestinal peptide, gastrin andglucagons. Kappa opioid receptor agonists such as fedotozine,ketocyclazocine, and compounds described in WO 03/097051 A2 can be usedwith or linked to the peptides of the invention. In addition, mu opioidreceptor agonists such as morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1) and loperamide can beused.

Tyr-Arg (kyotorphin) is a dipeptide that acts by stimulating the releaseof met-enkephalins to elicit an analgesic effect (J. Biol. Chem.262:8165, 1987). Kyotorphin can be used with or linked to the peptidesof the invention.

CCK receptor agonists such as caerulein from amphibians and otherspecies are useful analgesic agents that can be used with or linked tothe peptides of the invention.

Conotoxin peptides represent a large class of analgesic peptides thatact at voltage gated Ca channels, NMDA receptors or nicotinic receptors.These peptides can be used with or linked to the peptides of theinvention.

Peptide analogs of thymulin (FR 2830451) can have analgesic activity andcan be used with or linked to the peptides of the invention.

CCK (CCKa or CCKb) receptor antagonists, including loxiglumide anddexloxiglumide (the R-isomer of loxiglumide) (WO 88/05774) can haveanalgesic activity and can be used with or linked to the peptides of theinvention.

Other useful analgesic agents include 5-HT4 agonists such astegaserod/zelnorm and lirexapride. Such agonists are described in:EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, U.S. Pat. No.5,510,353, EP 507672 A1, EP 507672 B1, and U.S. Pat. No. 5,273,983.

Calcium channel blockers such as ziconotide and related compoundsdescribed in, for example, EP 625162B1, U.S. Pat. No. 5,364,842, U.S.Pat. No. 5,587,454, U.S. Pat. No. 5,824,645, U.S. Pat. No. 5,859,186,U.S. Pat. No. 5,994,305, U.S. Pat. No. 6,087,091, U.S. Pat. No.6,136,786, WO 93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1,U.S. Pat. No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No.6,054,429, WO 97/01351 A1, can be used with or linked to the peptides ofthe invention.

Various antagonists of the NK-1, NK-2, and NK-3 receptors (for a reviewsee Giardina et al. 2003 Drugs 6:758) can be can be used with or linkedto the peptides of the invention.

NK1 receptor antagonists such as: aprepitant (Merck & Co Inc),vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd),SR-14033 and related compounds described in, for example, EP 873753 A1,U.S. 20010006972 A1, U.S. 20030109417 A1, WO 01/52844 A1, can be usedwith or linked to the peptides of the invention.

NK-2 receptor antagonists such as nepadutant (Menarini Ricerche SpA),saredutant (Sanofi-Synthelabo), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc) can be used with or linked to the peptides of theinvention.

NK3 receptor antagonists such as osanetant (Sanofi-Synthelabo),talnetant and related compounds described in, for example, WO 02/094187A2, EP 876347 A1, WO 97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/11090,WO 95/28418, WO 97/19927, and Boden et al. (J Med Chem. 39:1664-75,1996) can be used with or linked to the peptides of the invention.

Norepinephrine-serotonin reuptake inhibitors such as milnacipran andrelated compounds described in WO 03/077897 A1 can be used with orlinked to the peptides of the invention.

Vanilloid receptor antagonists such as arvanil and related compoundsdescribed in WO 01/64212 A1 can be used with or linked to the peptidesof the invention.

Where the analgesic is a peptide and is covalently linked to a peptidedescribed herein the resulting peptide may also include at least onetrypsin or chymotrypsin cleavage site. When present within the peptide,the analgesic peptide may be preceded by (if it is at the carboxyterminus) or followed by (if it is at the amino terminus) a chymotrypsinor trypsin cleavage site that allows release of the analgesic peptide.

In addition to sialorphin-related peptides, analgesic peptides include:AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron,zicnotide, and substance P.

Methods of Treatment

The peptides of the invention can be used alone or in combinationtherapy for the treatment or prevention of cancer, pre-cancerousgrowths, or metastatic growths. For example, they can be used for theprevention or treatment of: colorectal/local metastasized colorectalcancer, gastrointestinal tract cancer, lung cancer, cancer orpre-cancerous growths or metastatic growths of epithelial cells, polyps,breast, colorectal, lung, ovarian, pancreatic, prostatic, renal,stomach, bladder, liver, esophageal and testicular carcinoma, carcinoma(e.g., basal cell, basosquamous, Brown-Pearce, ductal carcinoma, Ehrlichtumor, Krebs, Merkel cell, small or non-small cell lung, oat cell,papillary, bronchiolar, squamous cell, transitional cell, Walker),leukemia (e.g., B-cell, T-cell, HTLV, acute or chronic lymphocytic, mastcell, myeloid), histiocytonia, histiocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis, adenoma,adeno-carcinoma, adenofibroma, adenolymphoma, ameloblastoma,angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosingangioma, angiomatosis, apudoma, branchionia, malignant carcinoidsyndrome, carcinoid heart disease, carcinosarcoma, cementoma,cholangioma, cholesteatoma, chondrosarcoma, chondroblastoma,chondrosarcoma, chordoma, choristoma, craniopharyngioma, chrondrorna,cylindroma, cystadenocarcinoma, cystadenoma, cystosarconia phyllodes,dysgenninoma, ependymoma, Ewing sarcoma, fibroma, fibrosarcoma, giantcell tumor, ganglioneuroma, glioblastoma, glomangioma, granulosa celltumor, gynandroblastoma, hamartoma, hemangioendothelioma, hemangioma,hemangio-pericytoma, hemangiosarcoma, hepatoma, islet cell tumor, Kaposisarcoma, leiomyoma, leiomyosarcoma, leukosarcoma, Leydig cell tumor,lipoma, liposarcoma, lymphaugioma, lymphangiomyoma, lymphangiosarcoma,medulloblastoma, meningioma, mesenchymoma, mesonephroma, mesothelioma,myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, neurilemmoma,neuroma, neuroblastoma, neuroepithelioma, neurofibroma,neurofibromatosis, odontoma, osteoma, osteosarcoma, papilloma,paraganglioma, paraganglionia, nonchroinaffin, pinealoma, rhabdomyoma,rhabdomyosarcoma, Sertoli cell tumor, teratoma, theca cell tumor, andother diseases in which cells have become dysplastic, immortalized, ortransformed.

The peptides of the invention can be used alone or in combinationtherapy for the treatment or prevention of Familial AdenomatousPolyposis (FAP) (autosomal dominant syndrome) that precedes coloncancer, hereditary nonpolyposis colorectal cancer (HNPCC), and inheritedautosomal dominant syndrome.

For treatment or prevention of cancer, pre-cancerous growths andmetastatic growths, the peptides can be used alone or in combinationtherapy with radiation or chemotherapeutic agents, an inhibitor of acGMP-dependent phosphodiesterase or a selective cyclooxygenase-2inhibitor (a number of selective cyclooxygenase-2 inhibitors aredescribed in WO02062369, hereby incorporated by reference).

The peptides can be for treatment or prevention of inflammation. Thus,they can be used alone or in combination with inhibitors ofcGMP-dependent phosphodiesterase or a selective cyclooxygenase-2inhibitor for treatment of: organ inflammation, IBD (e.g, Crohn'sdisease, ulcerative colitis), asthma, nephritis, hepatitis,pancreatitis, bronchitis, cystic fibrosis, ischemic bowel diseases,intestinal inflammations/allergies, coeliac disease, proctitis,eosnophilic gastroenteritis, mastocytosis, and other inflammatorydisorders.

The peptides can also be used alone or in combination therapy to treator prevent insulin-related disorders, for example: II diabetes mellitus,hyperglycemia, obesity, disorders associated with disturbances inglucose or electrolyte transport and insulin secretion in cells, orendocrine disorders. They can be also used in insulin resistancetreatment and post-surgical and non-post surgery decrease in insulinresponsiveness.

The peptides can be used alone or in combination therapy to prevent ortreat respiratory disorders, including, inhalation, ventilation andmucus secretion disorders, pulmonary hypertension, chronic obstructionof vessels and airways, and irreversible obstructions of vessels andbronchi.

The peptides can be used in combination therapy with a phosphodiesteraseinhibitor (examples of such inhibitors can be found in U.S. Pat. No.6,333,354, hereby incorporated by reference).

The peptides can also be used alone or in combination therapy to preventor treat: retinopathy, nephropathy, diabetic angiopathy, and edemaformation

The peptides can also be used alone or in combination therapy to preventor treat neurological disorders, for example, headache, anxiety,movement disorders, aggression, psychosis, seizures, panic attacks,hysteria, sleep disorders, depression, schizoaffective disorders, sleepapnea, attention deficit syndromes, memory loss, and narcolepsy. Theymay also be used as a sedative.

The peptides and detectabley labeled peptides can be used as markers toidentify, detect, stage, or diagnosis diseases and conditions of thesmall intestine, including: Crohn's disease, colitis, inflammatory boweldisease, tumors, benign tumors, such as benign stromal tumors, adenoma,angioma, adenomatous (pedunculated and sessile) polyps, malignant,carcinoid tumors, endocrine cell tumors, lymphoma, adenocarcinoma,foregut, midgut, and hindgut carcinoma, gastroinstestinal stromal tumor(GIST), such as leiomyorna, cellular leiomyoma, leiomyoblastoma, andleiomyosarcoma, gastrointestinal autonomic nerve tumor, malabsorptionsyndromes, celiac diseases, diverticulosis, Meckel's diverticulurn,colonic diverticula, megacolon, Hirschsprung's disease, irritable bowelsyndrome, mesenteric ischemia, ischemic colitis, colorectal cancer,colonic polyposis, polyp syndrome, intestinal adenocarcinoma, Liddlesyndrome, Brody myopathy, infantile convulsions, and choreoathetosis

The peptides can be conjugated to another molecule (e.g, a diagnostic ortherapeutic molecule) to target cells bearing the GCC receptor, e.g.,cystic fibrosis lesions and specific cells lining the intestinal tract.Thus, they can be used to target radioactive moieties or therapeuticmoieties to the intestine to aid in imaging and diagnosing or treatingcolorectal/metastasized or local colorectal cancer and to deliver normalcopies of the p53 tumor suppressor gene to the intestinal tract.

The peptides can be used alone or in combination therapy to treaterectile dysfunction.

The peptides can be used alone or in combination therapy to treat innerear disorders, e.g., to treat Meniere's disease, including symptoms ofthe disease such as vertigo, hearing loss, tinnitus, sensation offullness in the ear, and to maintain fluid homeostasis in the inner ear.

The peptides can be used alone or in combination therapy to treatdisorders associated with fluid and sodium retention, e.g., diseases ofthe electrolyte-water/electrolyte transport system within the kidney,gut and urogenital system, congestive heart failure, hypertension,hypotension, liver cirrhosis, and nephrotic syndrome. In addition theycan be used to facilitate diuresis or control intestinal fluid.

The peptides can be used alone or in combination therapy to treatdisorders associated with chloride or bicarbonate secretion, e.g.,Cystic Fibrosis.

The peptides can be used alone or in combination therapy to treatdisorders associated with bile secretion. In addition, they can be usedto facilitate or control chloride and bile fluid secretion in the gallbladder.

The peptides can be used alone or in combination therapy to treatdisorders associated with liver cell regeneration.

1-57. (canceled)
 58. A method for treating a patient with agastrointestinal disorder, the method comprising administering to thepatient a composition comprising a polypeptide consisting of the aminoacid sequence Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr GlyCys Leu (SEQ ID NO:73), wherein one or two amino acids are substitutedby a D-isomer of the amino acid, wherein the gastrointestinal disorderis selected from the group consisting of a gastrointestinal motilitydisorder, irritable bowel syndrome, constipation, gastroesophagealreflux disease, functional heartburn, dyspepsia, functional dyspepsia,nonulcer dyspepsia, gastroparesis, chronic intestinal pseudo-obstructionand colonic pseudo-obstruction.
 59. (canceled)
 60. The method of claim58, wherein the gastrointestinal disorder is dyspepsia, functionaldyspepsia or nonulcer dyspepsia.
 61. The method of claim 60, wherein thegastrointestinal disorder is functional dyspepsia. 62-63. (canceled) 64.The method of claim 58, wherein the gastrointestinal disorder isirritable bowel syndrome.
 65. The method of claim 64, wherein theirritable bowel syndrome is constipation-predominant irritable bowelsyndrome (c-IBS).
 66. The method of claim 58, wherein thegastrointestinal disorder is constipation.
 67. The method of claim 66,wherein the constipation is idiopathic constipation.
 68. The method ofclaim 58, wherein the gastrointestinal disorder is gastroparesis.
 69. AThe method of claim 58, wherein the gastrointestinal disorder is agastrointestinal motility disorder.
 70. (canceled)
 71. A method fortreating a patient suffering from constipation-predominant irritablebowel syndrome, the method comprising orally administering to thepatient a composition comprising a polypeptide consisting of the aminoacid sequence Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr GlyCys Leu (SEQ ID NO: 73), wherein one or two amino acids are substitutedby the D-isomer of the amino acid.
 72. The method of claim 71, whereintwo amino acids are substituted by the D-isomer of the amino acid.
 73. Amethod for treating a patient suffering from constipation, the methodcomprising orally administering to the patient a composition comprisinga polypeptide consisting of the amino acid sequence Asn Asp Glu Cys GluLeu Cys Val Asn Val Ala Cys Thr Gly Cys Leu (SEQ ID NO: 73), wherein oneor two amino acids are substituted by the D-isomer of the amino acid.74. The method of claim 73, wherein two amino acids are substituted bythe D-isomer of the amino acid. 75-93. (canceled)
 94. The method ofclaim 58, wherein two amino acids are substituted by the D-isomer of theamino acid.